Regulation of nitric oxide release and biofilm development

ABSTRACT

The present invention relates generally to methods and compounds for regulating the release of nitric oxide in the vicinity of biofilm-forming microorganisms to regulate programmed cell death in the microorganisms and thereby promote dispersal of microorganism from biofilms and/or inhibit biofilm formation or development. More particularly, the invention relates to the use of compounds to provide spatial and temporal control over nitric oxide release.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/117,742, filed Apr. 10, 2014, which is a U.S. National PhaseApplication of PCT/AU12/00542, filed May 16, 2012, which claims benefitof Australian Patent Application No. 2011901872, filed May 16, 2011, thedisclosures of each of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The present invention relates generally to methods and compounds forregulating the release of nitric oxide in the vicinity ofbiofilm-forming microorganisms to regulate programmed cell death in themicroorganisms and thereby promote dispersal of microorganism frombiofilms and/or inhibit biofilm formation or development. Moreparticularly, the invention relates to the use of compounds to providespatial and temporal control over nitric oxide release.

BACKGROUND OF THE INVENTION

Biofilms are three dimensional microbial growth forms comprisingmicrobial communities and the extracellular matrix they produce.Biofilms are ubiquitous in nature, forming on any surface or at anyinterface where water or suitable fluid is available, or in suspension,for example as flocs or granules.

Biofilms are etiologic agents of a number of diseases and are associatedwith a variety of chronic infections in humans, forming on a variety ofsurfaces within the body, for example on surfaces in the respiratorytract and lungs (associated with cystic fibrosis and Legionnaire'sdisease), on surfaces of the ear (associated with otitis media), and onsurfaces of the heart and heart valves (associated with bacterialendocarditis). Biofilms offer increased protection to the microorganisminhabitants, for example in the form of substantially increasedresistance to antibiotics compared to planktonic cells and resistance tophagocytosis, which render biofilms very difficult to eradicate andexplains the severity and high level of persistence of biofilms and themorbidity associated with infections produced by biofilms. In the caseof cystic fibrosis, for example, a principal cause of respiratoryinfections is Pseudomonas aeruginosa, and P. aeruginosa biofilms on thesurface of the lungs in cystic fibrosis sufferers imparts a greaterdegree of antibiotic resistance and resistance to host immune defences.Consequently the major cause of chronic lung infections, and in turn ofmorbidity and mortality, in cystic fibrosis sufferers isbiofilm-associated P. aeruginosa.

Biofilms also readily form on medical equipment such as catheters andcannulas, and on implantable medical devices including stents andcontact lenses. Indeed many long term catheterization patients acquireinfections caused by biofilm-forming bacteria, and more generallybiofilms are responsible for a range of hospital acquired infections,adding considerable cost to health systems.

From a public health perspective, biofilms are important reservoirs ofpathogens in water systems such as drinking water, reservoirs, pipes andair-conditioning ducts. Biofilms also cause significant industrialdamage, causing, for example, fouling and corrosion in fluid processessuch as water distribution and treatment systems, pulp and papermanufacturing systems, heat exchange systems and cooling towers, andcontributing to the souring of oil in pipelines and reservoirs.

Biofilms are essentially multicellular microbial communities, theformation and development of which are dependent on variousmulticellular traits of the member organisms, such as cell-cellsignalling. Extracellular signalling systems such as quorum sensing areused by bacteria to assess cell density and initiate changes in geneexpression and phenotypes when sufficient concentrations of signallingmolecules are reached. This is associated with differential geneexpression, leading to the induction of, for example, virulence factorsand/or defence mechanisms, and with cell differentiation such thatbiofilm-associated cells become highly differentiated from planktoniccells.

As the cells within biofilms differentiate and biofilms mature, reducedmetabolic rates, the cellular expression of defence mechanisms and thereduced ability of antimicrobial agents to penetrate the biofilm resultsin increased antimicrobial resistance and make biofilms particularlydifficult to eradicate. Present biofilm control strategies typicallytarget the early stages of biofilm development and involve the use oftoxic antimicrobial agents. However such toxic agents can present theirown downstream problems, for example when used industrially due to theirrelease into the environment. Improved strategies for biofilm controlare clearly required.

Studies of P. aeruginosa, as well as other model biofilm formingbacteria, mixed species oral bioflims, and mixed species granularbiofilms in waste water treatment processes have shown that programmedcell death induces detachment and dispersal of cells from biofilms (see,for example, Hope et al., 2002 and Webb et al, 2003) and is a generalfeature of biofilm development. Inventors of the present invention havepreviously found that programmed cell death in biofilms is linked to theaccumulation of reactive oxygen and nitrogen species (RONS) withinbiofilm-forming organisms, and that programmed cell death and dispersalof cells from a biofilm into planktonic cells can be induced using low,non-toxic concentrations of nitric oxide generators or donors (seeco-pending WO 2006/125262, the disclosure of which is incorporatedherein by reference in its entirety).

The exploitation of this finding offers the prospect of noveltechnologies for the removal of biofilms in a broad range ofenvironments and settings, including medical, industrial andbioprocessing by exposing biofilms to nitric oxide to induce thedispersal of cells. However in some settings, in particular in humanhealth and medical applications, the uncontrolled and widespread releaseof nitric oxide may be associated with unacceptable side effects andtoxicity levels. Improving the stability of nitric oxide donors insolution also poses a challenge. Accordingly, there is a need for thedevelopment of effective mechanisms to regulate, spatially and/ortemporally, the release of nitric oxide such that this release can belocalised in the vicinity of a biofilm to thereby minimise side effectsand toxicity at other locations.

Now provided herein are compounds, methods and compositions forregulating the release of nitric oxide temporally and spatially and inturn providing novel mechanisms for promoting dispersal of cells frombiofilms and regulating biofilm development.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides a compound of theformula (I), or a salt

thereof:wherein T is a bond or a linker, R₂ and R₃ are organic residues and X isselected from the group consisting of:

wherein R₁ is an organic residue.

In one embodiment, the compound of the formula (I) has the followingstructure:

wherein R₁, R₂, R₃ and T are as defined above.

T may be a linker which is a bivalent hydrocarbon having between 1 and 6carbon atoms, for example —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)CH₂— or—CH₂CH₂CH₂CH₂—. In one embodiment T is CH₂.

R₁ may be a substituent that corresponds to a substituent attached tothe 7-NHC(O)— group of a cephalosporin antibiotic.

In one embodiment, R₁ is Y-aryl or Y-heteroaryl and Y is a bivalenthydrocarbon having between 1 and 4 carbon atoms. The aryl group may beselected from: phenyl, biphenyl, naphthyl, anthracenyl andphenanthrenyl, and the heteroaryl group may be a 5- or 6-membered ringwherein between 1 and 4 carbon atoms are replaced with nitrogen and/orsulfur atoms.

The heteroaryl group may be selected from: thienyl, tetrazolyl,imidazolyl, triazolyl and pyrrolyl.

In one embodiment, R₁ is selected from the group consisting of:—CH₂-phenyl, —CH₂-thienyl and —CH₂-tetrazolyl.

R₂ and R₃ may be independently selected from the group consisting of:C₁-C₁₀ alkyl, or alternatively R₂ and R₃ together with the nitrogen towhich they are attached may form a 5- or 6-membered ring which mayoptionally contain between 1 and 3 additional nitrogen atoms, and whichmay optionally be substituted with an aryl or heteroaryl group.

In an alternative embodiment R₂ and R₃ are independently selected fromC₁-C₆ alkyl, or alternatively R₂ and R₃, together with the nitrogen towhich they are attached, form a saturated 5- or 6-membered ring whichmay optionally contain 1 additional nitrogen atom, and which mayoptionally be substituted with a substituent selected from the groupconsisting of: pyrimidinyl and phenyl.

In another embodiment R₂ and R₃ are independently selected from C₁-C₆alkyl, or alternatively R₂ and R₃, together with the nitrogen to whichthey are attached, form a structure selected from the group consistingof:

In one embodiment the compound of formula (I) has a structure selectedfrom the group consisting of:

The compounds of formula (I) may further comprise an antibiotic compoundwhich is attached via the R₂ and/or R₃ substituent. The antibiotic maybe, for example, ciprofloxacin or N-desmethyl levofloxacin.

In a second aspect the invention provides a composition for promotingthe dispersal of microorganisms from a biofilm or inhibiting theformation and/or development of biofilms, the composition comprising acompound according to the first aspect.

The composition may further comprise one or more additional antibioticsor antimicrobial agents. In exemplary embodiments the one or moreadditional antibiotics may be selected from ceftazidime, tobramycin andciprofloxacin.

In a third aspect the invention provides a method for promotingdispersal of microorganisms from a biofilm, the method comprisingexposing the biofilm to an effective amount of a compound of the firstaspect or a composition of the second aspect.

In a fourth aspect the invention provides a method for inhibitingbiofilm formation and/or development, the method comprising exposingbiofilm-forming microorganisms to an effective amount of a compound ofthe first aspect or a composition of the second aspect.

In accordance with the fourth aspect, the compound or compositioncomprising the same may be coated, impregnated or otherwise contactedwith a surface or interface susceptible to biofilm formation. In oneembodiment the surface may be a surface of an implantable medicaldevice, prosthesis or medical or surgical equipment.

In accordance with the methods of the third and fourth aspects thebiofilm-containing or biofilm-forming microorganisms typically express aβ-lactamase or a transpeptidase. The β-lactamase may be encodedchromosomally or extrachromosomally and expression may be constitutiveor inducible. In particular embodiments the β-lactamase is apenicillinase. The biofilm or biofilm-forming microorganisms may beexposed to a β-lactam antibiotic prior to or concomitant with exposureto the compound or composition. The β-lactam antibiotic may induceproduction of extracellular β-lactamase in said biofilm-formingmicroorganisms. The β-lactam antibiotic may be provided in asubinhibitory, bacteriostatic or bacteriocidal concentration. Inparticular embodiments the β-lactam antibiotic is imipenem.

In particular embodiments of the third and fourth aspects the biofilmmay be on a bodily surface of a subject, internal or external to thesubject, and exposure of the biofilm or biofilm-forming microorganismsto the compound or composition may be via administration of the compoundor composition to the subject. Administration may be via any suitableroute depending on the nature and location of the biofilm orbiofilm-forming microorganisms.

Methods for promoting dispersal of or preventing formation of biofilmsmay comprise inducing differentiation events in microorganisms withinbiofilms which lead to dispersal or may comprise preventing induction ofdifferentiation events in microorganisms which lead to biofilmformation. Alternatively, or in addition, methods may compriseincreasing the sensitivity of a microorganism to antimicrobial agents.

In accordance with the above aspects and embodiments, the biofilm may besurface-associated or suspended. The suspended biofilm may be in theform of flocs or granules.

Typically in accordance with the above aspects and embodiments thebiofilm or biofilm-forming microorganisms are exposed to an effectiveamount of a compound or composition as defined here such that theconcentration of the nitric oxide donor or nitric oxide released andthus exposed to the biofilm or microorganisms is non-toxic to theenvironment or to the subject in which the biofilm or microrgansims arefound. For example, the concentration of nitric oxide may be in thenanomolar, micromolar or millimolar range. The nitric oxideconcentration may be, for example, from about 1 nM to about 500 μM.

The microorganisms present in the biofilm may be of a single species orof multiple species. The microorganisms within the biofilm or capable offorming a biofilm may comprise one or more species selected from, forexample, Pseudomonas spp., Pseudoalieromonas spp., Staphylococcus spp.,Streptococcus spp., Shigella spp., Mycobacterium spp., Enterococcusspp., Eschericia spp., Salmonella spp., Legionella spp., Haemophilusspp., Bacillus spp., Desulfovibrio spp., Shewanella spp., Geobacterspp., Klebsiella spp., Proteus spp., Aeromonas spp., Arthrobacter spp.,Micrococcus spp., Burkholderia spp., Serratia spp., Porphyromonas spp.,Fusobacterium spp. and Vibrio spp. In particular embodiments themicroorganism may be Pseudomonas aeruginosa, Staphylococcus epidermidis,Staphylococcus aureus, Mycobacterium tuberculosis, Escherichia coli,Bacillus licheniformis, Burkholderia cenocepacia, Serratia marcescens,Fusobacterium nucleatum, or Vibrio cholerae.

In particular embodiments the biofilm is on or within the body of asubject and may be associated with a disease or disorder suffered by thesubject. The disease or disorder may be, for example, cystic fibrosis,bacterial endocarditis, otitis media, Legionnaire's disease,tuberculosis or kidney stones.

Accordingly, in a fifth aspect there is provided a method for treatingor preventing a biofilm-associated infection, disease or disorder in asubject wherein the infection is caused by a microorganism capable offorming a biofilm, the method comprising administering to the subject aneffective amount of a compound of the first aspect or a composition ofthe second aspect.

The present invention also provides the use of a compound of the firstaspect for the manufacture of a composition for use in promotingdispersal of microorganisms from a biofilm or for inhibiting theformation or development of a biofilm.

The present invention also provides the use of a compound of the firstaspect for the manufacture of a medicament for treating or preventing abiofilm-associated infection, disease or disorder in a subject whereinthe infection is caused by a microorganism capable of forming a biofilm.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are described herein, by way ofnon-limiting example only, with reference to the following drawings.

FIG. 1. Amperometric characterization of nitric oxide release fromcephalosporin-3′-diazeniumdiolate free acids (Compounds 14 to 19).Arrows indicate addition of the following to a reaction vial containing10 mL Tris buffer at pH 7.0: (a) 10 μL of 100 mMcephalosporin-3′-diazeniumdiolate, (b) 10 μl 1 U/μl penicillinase, (c)20 μl 1 U/μl penicillinase, (d) 80 μL of 10 mM free radical scavengerPTIO.

FIG. 2. Amperometric characterization of nitric oxide release fromCompound 15. (a) Nitric oxide release in the presence of penicillinaseat varying pH. Arrows indicate addition of the following to a reactionvial containing 10 mL Tris buffer at pH 9.0 (thick line), 7.0 (dashedline) or 5.0 (dotted line): (i) 10 μL of 150 mM Compound 15, (ii) 5 μLof 0.1 U/μL penicillinase, (iii) 10 μL of 0.1 U/μL penicillinase, (iv)80 μL of 10 mM free radical scavenger PTIO. (b) Nitric oxide release inthe presence of β-lactamase-expressing P. aeruginosa (thick line) ornon-β-lactamase-expressing E. coli (dotted line) cell extracts. Arrowsindicate addition of the following to a reaction vial containing 10 mLTris buffer at pH 7.0: (i) 10 μL of 150 mM Compound 15, (ii) 100 μL cellextract, (iii) 200 μL cell extract, (iv) PTIO. Nitric oxide wasundetectable in the presence of penicillinase (1 U/mL) or arepresentative non-nitric oxide releasing cephalosporin cefalotin (150μM) either alone or from cefalotin/penicillinase mixtures (data notshown). The data is representative of at least three independentexperiments. (c) Nitric oxide release in the presence of supernatants ofP. aeruginosa cultures grown for 5 h in the absence of antibiotic thentreated for 1 h with extracellular β-lactamase-inducing imipenem a 0.5μg/ml (thick line) or ampicillin at 100 μg/ml (dotted line). Arrowsindicate addition of the following to a reaction vial containing 10 mLTris buffer at pH 7.0: (i) 10 μL of 150 mM Compound 15, (ii) 500 μLsupernatant, (iii) 500 μL supernatant, (iv) PTIO. Addition of 500 μl of100 μg/ml ampicillin to the vials with imipenem-treated cellssupernatant did not inhibit nitric oxide release from Compound 15. (datanot shown).

FIG. 3. Compound 21 induces a nitric oxide-dependent genetic response ina P. aeruginosa NSGFP reporter strain. NSGFP cells grown with or withoutsub-inhibitory ampicillin (50 μg/mL) were exposed to the spontaneousnitric oxide donor sodium nitroprusside (150 μM), compound 21 (150 μM),compound 21 (150 μM) plus penicillinase (0.2 U/mL), a representativenon-nitric oxide releasing cephalosporin cefalotin (150 μM) orpenicillinase (0.2 U/mL), or left untreated.

FIG. 4. Compound 21 (denoted as “DEA-CP” in the figure) induces rapiddispersal in P. aeruginosa biofilms. Exposure of preestablished biofilmsto Compound 21 for 10 min induces a concomitant decrease in biofilmbiomass (dark gray bars) and increase in planktonic biomass (light graybars). Error bars represent standard error; n=6.

FIG. 5. Compound 21 (denoted as “DEA-CP” in the figure) induces rapiddispersal in P. aeruginosa biofilms in a dose dependent manner (A).Biofilms of P. aeruginosa CF isolates strains grown of 24 h alsodisperse after exposure to Compound 21 for 10 min (light gray bars)compared to biofilms that were left untreated (dark gray bars) (B).Error bars represent standard error, n=2.

FIG. 6. Dose-dependent dispersal of P. aeruginosa biofilms by compounds21 (a) and 23 (b). P. aeruginosa biofilms were grown in microtiterplates with shaking at 37° C. and pre-treated with imipenem (0.5 μg/mL)for 1 h before exposing to various concentrations of compounds for 15mins. Remaining biofilm mass was quantified by crystal violet staining.

FIG. 7. Upon reaction with β-lactamase, Compound 21 induces a rapiddispersal response in P. aeruginosa biofilms. (A) Biofilms werequantified by crystal violet staining. (B) Pictures ofcrystal-violet-stained biofilms. (C) Planktonic cells were quantified byOD₆₀₀ measurement of the supernatant. Error bars indicate standarderrors (n=2). In these experiments, biofilm dispersal is supported bythe increase in planktonic OD readings that correspond with decreases incrystal violet staining of biofilms.

FIG. 8. Compound 21 induces rapid dispersal (10 min after treatment) inbiofilms of various Gram-negative bacteria: (A) Escherichia coli, (B)Vibrio cholerae, (C) Serratia marcescens; and Gram-positive bacteria:(D) Staphylococcus aureus.

FIG. 9. Compound 21 induces dispersal of P. aeruginosa PAO1 biofilmsgrown in glass microfermentors under continuous flow conditions. After24 h growth, OD₆₀₀ measurements of the biofilm effluent showed asubstantial increase in released cells after addition of compound 21,while the amount of released cells from untreated biofilms remainedunchanged. Arrow indicates addition of compound. Data is representativeof two independent experiments.

FIG. 10. Planktonic growth inhibition by cephalothin, DEA and Compound21 (denoted as “DEA-CP” in the figure) in wild type P. aeruginosa. Thegraph only shows data for concentrations at 4 mM and 16 mM; lowerconcentrations had no effect and higher concentrations completelyinhibited growth for all 3 compounds. Error bars indicate standarderror; n=2.

FIG. 11. Effect of combined treatments of Compound 21 (denoted as“DEA-CP” in the figure) an antibiotic on viability of P. aeruginosabiofilm and planktonic cells. Preestablished biofilms grown in wells ofmicrotiter plates were exposed, in the presence or absence of Compound21, to ceftazidime or left untreated. After 1 h (A, B) or 2 h (C, D)treatments, supernatants were collected and planktonic CFU wereenumerated (B, D); biofilm bacteria were resuspended in buffer andbiofilm CFU enumerated (A, C). Error bars indicate standard error; n=2.

DETAILED DESCRIPTION

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

In the context of this specification, the term “about” is understood torefer to a range of numbers that a person of skill in the art wouldconsider equivalent to the recited value in the context of achieving thesame function or result.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

As used herein the term “antimicrobial agent” refers to any agent that,alone or in combination with another agent such as an antibiotic, iscapable of killing or inhibiting the growth of one or more species ofmicroorganisms.

As used herein the term “biofilm” refers to any three-dimensional,matrix-encased microbial community displaying multicellularcharacteristics. Accordingly, as used herein, the term biofilm includessurface-associated biofilms as well as biofilms in suspension, such asflocs and granules. Biofilms may comprise a single microbial species ormay be mixed species complexes, and may include bacteria as well asfungi, algae, protozoa, or other microorganisms.

The term “biofilm-forming microorganism” refers to any microorganismthat is capable of forming biofilms, either single species or mixedspecies biofilms.

As used herein the term “dispersal” as it relates to a biofilm andmicroorganisms making up a biofilm means the process of detachment andseparation of cells and a return to a planktonic phenotype or behaviourof the dispersing cells.

As used herein the term “effective amount” includes within its meaning anon-toxic but sufficient amount of an agent to provide the desiredeffect. The exact amount required will vary from subject to subjectdepending on factors such as the species of microorganisms beingtreated, the extent, severity and/or age of the biofilm being treated,whether the biofilm is surface-associated or suspended, the particularagent(s) being administered and the mode of administration and so forth.Thus, it is not possible to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” may bedetermined by one of ordinary skill in the art using only routineexperimentation.

As used herein the term “exposing” means generally bringing into contactwith. Typically direct exposure refers to administration of the agent tothe microorganism or biofilm to be treated or otherwise bringing themicroorganism or biofilm into contact with the agent itself Typicallyindirect exposure refers to the administration of a precursor of theactive agent or a compound or molecule capable of generating, eithersolely or in reaction with other compounds or molecules, the activeagent to the microorganism or biofilm or otherwise bringing themicroorganism or biofilm into contact therewith. Thus, a microorganismor biofilm may be exposed to compound or composition as defined hereindirectly or indirectly. Further, a microorganism or biofilm may beexposed to nitric oxide released from a compound directly or indirectly.In the context of the present disclosure, indirectly “exposing” abiofilm or microorganisms to a compound or composition as defined hereinalso includes the administration of the compound or composition to asubject in or on which the biofilm or microorganisms reside. Thus, inthe present disclosure the terms “exposing”, “administering” and“delivering” and variations thereof may, in some contexts, be usedinterchangeably.

The term “inhibiting” and variations thereof such as “inhibition” and“inhibits” as used herein in relation to biofilms means complete orpartial inhibition of biofilm formation and/or development and alsoincludes within its scope the reversal of biofilm development orprocesses associated with biofilm formation and/or development. Further,inhibition may be permanent or temporary. The inhibition may be to anextent (in magnitude and/or spatially), and/or for a time, sufficient toproduce the desired effect Inhibition may be prevention, retardation,reduction or otherwise hindrance of biofilm formation or development.Such inhibition may be in magnitude and/or be temporal or spatial innature. Further, such inhibition may be direct or indirect. By indirectinhibition is meant that the agent may effect the expression or activityof molecules which in turn regulate biofilm formation or development.

As used herein the term “programmed cell death” means a developmentalevent within a biofilm that occurs at defined stages and causesautolysis, cellular differentiation and the development ofsubpopulations of cells with specific phenotypes.

Similarly, the term “promoting” and variations thereof such as“promotion” and “promotes” as used herein in the context of promotingthe dispersal of microorganisms from a biofilm also complete or partialpromotion of dispersal, which may be permanent or temporary, to anextent (in magnitude and/or spatially), and/or for a time, sufficient toproduce the desired effect. Such promotion may be direct or indirect.

As used herein the term “surface” includes both biological surfaces andnon-biological surfaces. Biological surfaces typically include surfacesboth internal (such as organs, tissues, cells, bones and membranes) andexternal (such as skin, hair, epidermal appendages, seeds, plantfoliage) to an organism. Biological surfaces also include other naturalsurfaces such as wood or fibre. A non-biological surface may be anyartificial surface of any composition that supports the establishmentand development of a biofilm. Such surfaces may be present in industrialplants and equipment, and include medical and surgical equipment andmedical devices, both implantable and non-implantable. Further, for thepurposes of the present disclosure, a surface may be porous (such as amembrane) or non-porous, and may be rigid or flexible.

As used herein the terms “treating”, “treatment”, “preventing” and“prevention” refer to any and all uses which remedy a condition orsymptoms, prevent the establishment of a condition or disease, orotherwise prevent, hinder, retard, or reverse the progression of acondition or disease or other undesirable symptoms in any waywhatsoever. Thus the terms “treating” and “preventing” and the like areto be considered in their broadest context. For example, treatment doesnot necessarily imply that a patient is treated until total recovery.

In the context of this specification, the term “C₁-C₂₀ alkyl” is takento include straight chain and branched chain monovalent saturatedhydrocarbon groups having 1 to 20 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like.

In the context of this specification, the term “C₁-C₁₀ alkyl” is takento include straight chain and branched chain monovalent saturatedhydrocarbon groups having 1 to 10 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl,hexyl, heptyl, octyl and the like.

In the context of this specification, the term “C₁-C₆ alkyl” is taken toinclude straight chain and branched chain monovalent saturatedhydrocarbon groups having 1 to 6 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl and the like.

In the context of this specification, the term “C₂-C₂₀ alkenyl” is takento include straight chain and branched chain monovalent hydrocarbonradicals having 2 to 20 carbon atoms and at least one carbon-carbondouble bond, such as vinyl, propenyl, 2-methyl-2-propenyl, butenyl,pentenyl, hexenyl, heptenyl, undecenyl and the like.

In the context of this specification, the term “C₂-C₁₀ alkenyl” is takento include straight chain and branched chain monovalent hydrocarbonradicals having 2 to 10 carbon atoms and at least one carbon-carbondouble bond, such as vinyl, propenyl, 2-methyl-2-propenyl, butenyl,pentenyl and the like.

In the context of this specification, the term “C₂-C₆ alkenyl” is takento include straight chain and branched chain monovalent hydrocarbonradicals having 2 to 6 carbon atoms and at least one carbon-carbondouble bond, such as vinyl, propenyl, 2-methyl-2-propenyl and the like.

In the context of this specification, the term “C₂-C₂₀ alkynyl” is takento include straight chain and branched chain monovalent hydrocarbonradicals having 2 to 20 carbon atoms and at least one carbon-carbontriple bond, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl,undecynyl and the like.

In the context of this specification, the term “C₂-C₁₀ alkynyl” is takento include straight chain and branched chain monovalent hydrocarbonradicals having 2 to 10 carbon atoms and at least one carbon-carbontriple bond, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl andthe like.

In the context of this specification, the term “C₂-C₆ alkynyl” is takento include straight chain and branched chain monovalent hydrocarbonradicals having 2 to 6 carbon atoms and at least one carbon-carbontriple bond, such as ethynyl, propynyl, butynyl and the like.

In the context of this specification, the term “aryl” is taken toinclude monovalent aromatic radicals having between 6 and 30 carbonatoms, for example phenyl, biphenyl, naphthyl, anthracenyl,phenanthrenyl and the like.

In the context of this specification, the term “heteroaryl” is taken toinclude monovalent aromatic radicals having between 4 and 25 atoms,wherein 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 or 2 atoms areheteroatoms selected from nitrogen, oxygen and sulfur, for examplefuranyl, quinazolinyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl,benzopyranyl, benzooxazolyl, benzimidazolyl, pyrazolyl, tetrazolyl,oxazolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, quinolizinyl, pyranyl,isothiazolyl, thiazolyl, thienyl, imidazolyl, pyrazinyl, pyridazinyl,pyrimidinyl, isothiazolyl, pyridyl, triazolyl, benzothienyl, pyrrolyl,benzothiazolyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl,azepinyl, acridinyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl,benzofuryl, purinyl, benzimidazolyl, triazinyl and the like.

In the context of this specification, the terms “halo” and “halogen” maybe used interchangeably and are taken to include fluoro, chloro, bromoand iodo.

In the context of this specification, the term “C₃-C₇ cycloalkyl” istaken to include cyclic alkyl groups having between 3 and 7 carbonatoms, for example cyclobutyl, cyclohexyl and the like.

In the context of this specification, the term “C₅-C₇ cycloalkyl” istaken to include cyclic alkyl groups having between 5 and 7 carbonatoms, for example cyclopentyl and the like.

In the context of this specification, the term “C₃-C₇ cycloalkenyl” istaken to include cyclic hydrocarbon groups having between 3 and 7 carbonatoms and at least one carbon-carbon double bond, for examplecyclopropenyl, cyclopentenyl, cyclohexenyl and the like.

In the context of this specification, the term “C₁-C₃ alkylene” is takento include bivalent hydrocarbon radicals having between 1 and 3 carbonatoms, for example methylene and ethylene.

β-lactamases are enzymes produced by bacteria in defence againstβ-lactam antibiotics. Many biofilm-forming microorganisms such asPseudomonas aeruginosa are capable of producing β-lactamases and producelarge quantities of these enzymes during biofilm formation and withinbiofilms, assisting to render β-lactam antibiotics ineffective ineradicating biofilms. As described and exemplified herein the presentinventors have now found that the coupling of a β-lactam antibiotic orthe β-lactam ring-containing core of a β-lactam antibiotic orantimicrobial agent to a nitric oxide donor compound enables thetargeted delivery of effective concentrations of nitric oxide and thespatial and temporal control over nitric oxide release upon exposure tobiofilms and biofilm-forming microorganisms to promote dispersal ofmicroorganisms from biofilms. As exemplified herein the conjugatecompounds of the present disclosure are stable in solution andeffectively make nitric oxide available to microorganisms in biofilms,being demonstrated to induce the rapid dispersal of P. aeruginosabiofilms after only 10 minutes exposure at concentrations in themicromolar range.

Accordingly, provided herein are conjugate compounds, compositionscomprising the same and uses thereof, wherein the conjugates comprise aβ-lactam antibiotic or β-lactam ring-containing antimicrobial agent, ora derivative thereof, complexed with a nitric oxide donor compound. Suchconjugates are stable in solution and act as nitric oxide prodrugsenabling the delivery of low, non-toxic concentrations of nitric oxideto desired sites to promote the dispersal of microorganisms frombiofilms and inhibit the formation and/or development of biofilms.

In one aspect the present invention provides a compound of the formula(I), or a salt thereof:

wherein T is a bond or a linker, R₂ and R₃ are organic residues and X isselected from the group consisting of:

wherein R₁ is an organic residue.

The compounds of formula (I) may have one or more chiral centres. Thepresent invention includes all enantiomers and diastereoisomers, as wellas mixtures thereof in any proportions. The invention also extends toisolated enantiomers or pairs of enantiomers. In embodiments of theinvention X is selected from the group consisting of:

Also within the scope of the compounds of formula (I) are salts,including pharmaceutically acceptable salts. Salts of the compounds offormula (I) may be prepared by conventional methods known to thoseskilled in the art. For example, base-addition salts may be prepared byreacting the compounds of formula (I) with a suitable base. Examples ofsuch salts include alkali metal salts, such as lithium, potassium andsodium, and alkali earth metal salts, such as calcium, magnesium andbarium. Additional basic salts include, but are not limited to,ammonium, copper, iron, manganese and zinc salts. Acid addition saltsmay be prepared by reacting the compounds of formula (I) with organic orinorganic acids. Examples of such salts include HCl, HBr and HI salts,salts of other mineral acids such as sulfate, nitrate, phosphate and thelike, alkyl and monoarylsulfonates such as ethanesulfonate,toluenesulfonate and benzene sulfonate, and salts of other organicacids, such as acetate, trifluoroacetate, tartrate, maleate, citrate,benzoate, ascorbate and the like. Compounds of the formula (I) may alsobe quaternised by reaction with compounds such as (C₁-C₄)alkyl halides,for example, methyl, ethyl, isopropyl and butyl halides.

T may be a bivalent linker having between 1 and 20 carbon atoms. In oneembodiment, T is a bivalent hydrocarbon linker having between 1 and 20carbon atoms, 1 and 15 carbon atoms, or between 1 and 10 carbon atoms.In another embodiment, T is selected from the group consisting of:—CH₂—, —CH₂CH₂—, —CH(CH₃)CH₂—, —CH₂CH₂CH₂— and —CH₂CH₂CH₂CH₂—.

R₁ may be a substituent corresponding to a substituent attached to the7-NHC(O)— group of a cephalosporin antibiotic. For example, R₁ may be asubstituent corresponding to a substituent attached to the 7-NHC(O)—group of any of the following: cefmatilen, cephaloram, cefazolin,cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium,cefaloridine, cefalotin, cefapirin, cefatrizine, cefazedone, cefazaflur,cefradine, cefroxidine, ceftezole, cefachlor, cefamandole, cefminox,cefonicid, ceforanide, cefotiam, cefprozil, cefbuperazone, cefuroxime,cefuzonam, cephamycin, cefotetan, cefmetazole, flomoxef, cefixime,ceftriaxone, ceftazidine, cefoperazone, cefcapene, cefdaloxime,cefdinir, cefditoran, cefetamet, cefmenoxime, cefodizime, cefotaxime,cefpimizole, cefpirimide, cefpodoxime, cefsulodin, cefteram, ceftibuten,ceftiolene, ceftizoxime, latamoxef, cefepine, cefozopram, cefpirome,cefquinome, ceftobiprole, ceftaroline fossamil and ceftiofur. It is tobe noted that R₁ may be a substituent corresponding to a substituentattached to the 7-NHC(O)— group of any clinically useful cephalosporinantibiotic.

In one embodiment R₁ is selected from the group consisting of:

The above noted groups correspond to the substituents attached to the7-NHC(O)— group of the specific cephalosporin antibiotics recited above.

In an alternative embodiment, R₁ is of the formula —Y-aryl, wherein Y isa bivalent hydrocarbon having between 1 and 6 carbon atoms. In oneembodiment, Y is a straight chain or branched chain hydrocarbon havingbetween 1 and 4 carbon atoms and aryl is phenyl or naphthyl.

In another embodiment, R₁ is Y-aryl or Y-heteroaryl where Y is abivalent hydrocarbon having 1 to 4 carbon atoms and aryl and heteroarylare optionally substituted.

In a further embodiment, R₁ is Y-aryl or Y-heteroaryl where Y is abivalent hydrocarbon having 1 to 4 carbon atoms, and the aryl group isselected from: phenyl, biphenyl, naphthyl, anthracenyl andphenanthrenyl, and the heteroaryl group is a 5- or 6-membered ringwherein between 1 and 4 carbon atoms are replaced with nitrogen and/orsulfur atoms, and wherein the aryl and heteroaryl groups may optionallybe substituted with one or more substituents selected from: C₁₋₆ alkyl,halo, amino, hydroxyl, methoxy and ethoxy.

In a further embodiment, R₁ is Y-aryl or Y-heteroaryl where Y is abivalent hydrocarbon having 1 to 4 carbon atoms, and the aryl group isselected from: phenyl, biphenyl, naphthyl, anthracenyl andphenanthrenyl, and the heteroaryl group is a 5- or 6-membered ringwherein between 1 and 4 carbon atoms are replaced with nitrogen and/orsulfur atoms.

In a further embodiment, R₁ is Y-aryl or Y-heteroaryl where Y is abivalent hydrocarbon having 1 to 3 carbon atoms, and the aryl group isselected from: phenyl, biphenyl and naphthyl, and the heteroaryl groupis selected from thienyl, tetrazolyl, imidazolyl, triazolyl, thiazolyl,isothiazolyl and pyrrolyl.

In a further embodiment, R₁ is Y-aryl or Y-heteroaryl where Y is abivalent hydrocarbon having 1 or 2 carbon atoms, and the aryl group isselected from: phenyl, biphenyl and naphthyl, and the heteroaryl groupis selected from thienyl, tetrazolyl, imidazolyl, triazolyl, thiazolyl,isothiazolyl and pyrrolyl.

In a further embodiment, R₁ is Y-aryl or Y-heteroaryl where Y is —CH₂—,and the aryl group is selected from: phenyl, biphenyl and naphthyl, andthe heteroaryl group is selected from thienyl, tetrazolyl, imidazolyl,triazolyl, thiazolyl, isothiazolyl and pyrrolyl.

In one embodiment, R₁ is selected from the group consisting of:—CH₂-phenyl, —CH₂-thienyl and —CH₂-tetrazolyl.

R₂ and R₃ may independently be selected from: hydrogen, C₃-C₇cycloalkyl, C₃-C₇ cycloalkenyl, (CH₂)_(p)OC(O)PhOC(O)C₁-C₆alkyl,(CH₂)_(p)OC(O)APhC₁-C₆alkyl, branched or straight chain C₁-C₂₀ alkyl,branched or straight chain C₂-C₂₀ alkenyl, branched or straight chainC₂-C₂₀ alkynyl, wherein the alkyl, alkenyl or alkynyl chains mayoptionally be interrupted by one or more groups/heteroatoms selectedfrom O, S, NH, NH₂ ⁺, and wherein the alkyl, alkenyl or alkynyl groupsmay optionally be substituted by one or more substituents selected fromthe group consisting of: halogen, cyano, COOH, (CH₂)_(p)C(O)OC₁-C₆alkyl,C(O)OC₁-C₆alkenyl, SO₃H, SO₂halogen, SO₂NH₂, NH₂, NH₃ ⁺, OH, SH,OC₁-C₆alkyl, OC₂-C₆alkenyl, OC₂-C₆alkynyl, aryl and heteroaryl, oralternatively R₂ and R₃, together with the nitrogen to which they areattached, form a 4-, 5-, 6-, 7-or 8-membered ring which may optionallycontain 1, 2, 3, 4, 5 or 6 additional nitrogen atoms and may besaturated, unsaturated or partially unsaturated, and wherein the 4-, 5-,6-, 7- or 8-membered ring may optionally be substituted by one or moresubstituents selected from the group consisting of:—C(O)C₁-C₃alkylene-naphthyl-OC₁-C₆alkyl, —C(O)C₁-C₃alkylene-Ph—C(O)—Ph,—C(O)CH₂O(CH₂)_(p)OCH₃, —C(O)OPhNO₂, —C(O)OPhNH₂,—C(O)O(CH₂)_(p)Chalogen₃, —C(O)O(CH₂O)_(p)CH₃, C₁-C₆-alkyl,C₂-C₆alkenyl, C₂-C₆alkynyl, —C(O)C₁-C₆alkyleneCOOC₁-C₆alkyl,—C(O)C₁-C₃alkylenePhC₁-C₆alkyl, —C(O)O-pyrrolidinyl-2,5-dione,—C(O)C₁-C₃alkylenePhC₁-C₆alkyl, —C(O)(CH₂)_(p)OC₁-C₆alkyl,—C(O)O(CH₂)_(p)halogen, —C(O)O(CH₂)_(p)Ph, —(CH₂)_(p)SH,—SO₂naphthyl-NC₁-C₆alkyl, —C(O)ONC₁-C₆alkyl, —(CH₂)_(p)OH, —C(O)PhOAc,—C(O)(CH₂)_(p)NHC(O)C₁-C₆alkyl, —C(O)NH₂, —C(O)M, —C(O)NR₄R₅,—(CH₂)_(p)CH(OH)CHOH, halogen, cyano, —COOH, —C(O)O(CH₂)_(p)Ph,—C(O)OC₁-C₆alkyl, —C(O)OC₂-C₆alkenyl, —C(O)OC₂-C₆alkynyl,—C(O)SC₁-C₆alkyl, —C(O)SC₂-C₆alkenyl, —C(O)SC₂-C₆alkynyl,—C(O)C₁-C₆alkyl, SO₃H, SO₂halogen, SO₂phenyl, SO₂NH₂, SO₂NR₄R₅,SO₂PhNHCOC₁-C₆alkyl, NH₂, OH, SH, OC₁-C₆alkyl, OC₂-C₆alkenyl,OC₂-C₆alkynyl, aryl and heteroaryl, and wherein A is a bivalenthydrocarbon radical having between 1 and 4 carbon atoms, p is a numberbetween 0 and 4, R₄ and R₅ independently represent C₁-C₆alkyl and M ispyridyl, pyrimidinyl, pyrazinyl, phenyl or triazinyl.

In an alternative embodiment, R₂ and R₃ may independently be selectedfrom: hydrogen, C₅-C₇ cycloalkyl, (CH₂)_(p)OC(O)PhOC(O)C₁-C₆alkyl,(CH₂)_(p)OC(O)APhC₁-C₆alkyl, branched or straight chain C₁-C₁₀ alkyl,branched or straight chain C₂-C₁₀ alkenyl and branched or straight chainC₂-C₁₀ alkynyl, wherein the alkyl, alkenyl or alkynyl chains mayoptionally be interrupted by between one and three groups/heteroatomsselected from O, S, NH and NH₂ ⁺, and wherein the alkyl, alkenyl oralkynyl chains may optionally be substituted by between one and sixsubstituents selected from the group consisting of: halogen, phenyl,ethoxy, methoxy, propoxy, COOH, (CH₂)_(p)COOC₁-C₄alkyl, NH₂, NH₃ ⁺, OHand SH, and wherein A is a bivalent hydrocarbon radical having between 1or 2 carbon atoms and p is 0, 1 or 2.

In another embodiment, R₂ and R₃ may independently be selected from:hydrogen, C₅-C₇ cycloalkyl, branched or straight chain C₁-C₁₀ alkyl,branched or straight chain C₂-C₁₀ alkenyl, branched or straight chainC₂-C₁₀ alkynyl, wherein the alkyl, alkenyl or alkynyl chains mayoptionally be interrupted by between one and three groups selected fromO, NH and NH₂ ⁺, and wherein the alkyl, alkenyl or alkynyl chains mayoptionally be substituted by between one and four substituents selectedfrom the group consisting of: halogen, phenyl, methoxy, COOH,CH₂COOC₁-C₄alkyl, NH₂ and NH₃ ⁺.

In yet another embodiment, R₂ and R₃ may independently be selected from:hydrogen, cyclohexyl, branched or straight chain C₁-C₁₀ alkyl orbranched or straight chain C₂-C₁₀ alkenyl, wherein the alkyl or alkenylchains may optionally be interrupted by one or two groups selected fromNH and NH₂ ⁺, and wherein the alkyl, alkenyl or alkynyl chains mayoptionally be substituted by between one and three substituents selectedfrom the group consisting of: phenyl, methoxy, COOH, NH₂ and NH₃ ⁺.

In still a further embodiment, R₂ and R₃, together with the nitrogen towhich they are attached, form a 4-, 5-, 6- or 7-membered ring which mayoptionally contain 1, 2, 3, 4 or 5 additional nitrogen atoms and may besaturated, unsaturated or partially unsaturated, and wherein the 4-, 5-,6- or 7-membered ring may optionally be substituted by one or moresubstituents selected from the group consisting of: SO₂NMe₂, SO₃H,SO₂halogen, SO₂NH₂, —C(O)O(CH₂)_(p)Ph, —C(O)Me, —C(O)pyridyl,—(CH₂)_(p)OH, —C(O)NH₂, —COOH, —C(O)NMe₂, —C(O)NEt₂, phenyl, naphthyl,pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolidinyl, imidazolyl,C₁-C₆-alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, —C(O)OC₁-C₆alkyl,—C(O)OC₂-C₆alkenyl, —C(O)OC₂-C₆alkynyl, —C(O)O(CH₂)_(p)Ph, —(CH₂)_(p)SH,halogen, SO₂PhNHCOC₁-C₆alkyl, NH₂, SH, OC₁-C₆alkyl, and wherein p is anumber between 0 and 2,

In another embodiment, R₂ and R₃, together with the nitrogen to whichthey are attached, form a 5-, 6-, or 7-membered ring which mayoptionally contain 1, 2 or 3 additional nitrogen atoms and may besaturated, unsaturated or partially unsaturated, and wherein the 5-, 6-,or 7-membered ring may optionally be substituted by between one and foursubstituents selected from the group consisting of: SO₂NMe₂, SO₂NH₂,—COO(CH₂)_(p)Ph—C(O)Me, —C(O)pyridyl, —(CH₂)_(p)OH, —C(O)NH₂, —COOH,—C(O)NMe₂, —C(O)NEt₂, phenyl, pyridinyl, pyrimidinyl, pyrazinyl,triazinyl, pyrrolidinyl, imidazolyl, C₁-C₆-alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, —C(O)OC₁-C₆alkyl, —C(O)OC₂-C₆alkenyl, —C(O)OC₂-C₆alkynyl,—C(O)O(CH₂)_(p)Ph, —(CH₂)_(p)SH, halogen, NH₂, SH, OC₁-C₆alkyl, p is anumber between 0 and 2,

In a further embodiment, R₂ and R₃, together with the nitrogen to whichthey are attached, form a saturated 5-, 6-, or 7-membered ring which mayoptionally contain 1, 2 or 3 additional nitrogen atoms, and wherein the5-, 6-, or 7-membered ring may optionally be substituted by between oneand three substituents selected from the group consisting of: SO₂NMe₂,SO₂NH₂, —C(O)Me, —C(O)pyridyl, —(CH₂)_(p)OH, —C(O)NH₂, —COOH, —C(O)NMe₂,—C(O)NEt₂, phenyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl,pyrrolidinyl, imidazolyl, C₁-C₆-alkyl, C₂-C₆alkenyl, —C(O)OC₁-C₆alkyl,—C(O)OC₂-C₆alkenyl, halogen, NH₂, SH, OC₁-C₆alkyl, p is a number between0 and 2.

In a further embodiment R₂ and R₃ are independently selected from C₁-C₁₀alkyl, or alternatively R₂ and R₃, together with the nitrogen to whichthey are attached, form a 5- or 6-membered ring which may optionallycontain between 1 and 3 additional nitrogen atoms, and which mayoptionally be substituted with an aryl or heteroaryl group.

In a further embodiment R₂ and R₃ are independently selected from theC₁-C₁₀ alkyl, or alternatively R₂ and R₃, together with the nitrogen towhich they are attached, form a 5- or 6-membered saturated ring whichmay optionally contain between 1 and 3 additional nitrogen atoms, andwhich may optionally be substituted with an aryl or heteroaryl group.

In another embodiment R₂ and R₃ are independently selected from C₁-C₆alkyl, or alternatively R₂ and R₃, together with the nitrogen to whichthey are attached, form a saturated 5- or 6-membered ring which mayoptionally contain 1 or 2 additional nitrogen atoms, and which mayoptionally be substituted with a substituent selected from the groupconsisting of: pyrimidinyl, naphthyl, phenyl, pyrazinyl, triazinyl,triazolyl, imidazolyl, tetrazolyl and pyrrolyl.

In another embodiment R₂ and R₃ are independently selected from C₁-C₆alkyl, or alternatively R₂ and R₃, together with the nitrogen to whichthey are attached, form a saturated 5- or 6-membered ring whichoptionally contains 1 additional nitrogen atom, and which may optionallybe substituted with a substituent selected from the group consisting of:pyrimidinyl, naphthyl, phenyl, pyrazinyl, triazinyl, triazolyl,imidazolyl, tetrazolyl and pyrrolyl.

In another embodiment R₂ and R₃ are independently selected from C₁-C₆alkyl, or alternatively R₂ and R₃, together with the nitrogen to whichthey are attached, form a saturated 5- or 6-membered ring whichoptionally contains 1 additional nitrogen atom, and which may optionallybe substituted with a substituent selected from the group consisting of:pyrimidinyl, phenyl, pyrazinyl and triazinyl.

In yet another embodiment R₂ and R₃ are independently selected frommethyl, ethyl or propyl, or alternatively R₂ and R₃, together with thenitrogen to which they are attached, form a saturated 5- or 6-memberedring which optionally contains 1 additional nitrogen atom, and which mayoptionally be substituted on the additional nitrogen with a substituentselected from the group consisting of: pyrimidinyl, phenyl, pyrazinyland triazinyl.

In still a further embodiment R₂ and R₃ are independently selected fromC₁-C₆ alkyl, or alternatively R₂ and R₃, together with the nitrogen towhich they are attached, form a structure selected from the groupconsisting of:

In a further embodiment R₂ and R₃ are independently selected from C₁-C₆alkyl.

In yet another embodiment, R₂ and R₃ are independently selected from thegroup consisting of: methyl, ethyl, propyl and isopropyl.

In particular embodiments, R₂ and R₃, together with the nitrogen towhich they are attached form the following structures:

In an alternative embodiment T is CH₂, R₁ is Y-aryl or Y-heteroarylwhere Y is a bivalent hydrocarbon having 1 to 4 carbon atoms and aryland heteroaryl are optionally substituted with between 1 and 3substituents selected from the group consisting of: C₁-C₆ alkyl, halo,amino and OC₁-C₆ alkyl, and R₂ and R₃ are independently selected fromC₁-C₁₀ alkyl, or alternatively R₂ and R₃, together with the nitrogen towhich they are attached, form a 5- or 6-membered ring which mayoptionally contain between 1 and 3 additional nitrogen atoms, and whichmay optionally be substituted with an aryl or heteroaryl group.

In an alternative embodiment T is CH₂, R₁ is Y-aryl or Y-heteroarylwhere Y is a bivalent hydrocarbon having 1 to 4 carbon atoms and aryland heteroaryl are optionally substituted, and R₂ and R₃ areindependently selected from C₁-C₁₀ alkyl, or alternatively R₂ and R₃,together with the nitrogen to which they are attached, form a 5- or6-membered ring which may optionally contain between 1 and 3 additionalnitrogen atoms, and which may optionally be substituted with an aryl orheteroaryl group.

In a further embodiment T is CH₂, R₁ is Y-aryl or Y-heteroaryl where Yis a bivalent hydrocarbon having 1 to 4 carbon atoms, and the aryl groupis selected from: phenyl, biphenyl, naphthyl, anthracenyl andphenanthrenyl, and the heteroaryl group is a 5- or 6-membered ringwherein between 1 and 4 carbon atoms are replaced with nitrogen and/orsulfur atoms, and wherein the aryl and heteroaryl groups may optionallybe substituted with one or more substituents selected from: C₁₋₆ alkyl,halo, amino, hydroxyl, methoxy and ethoxy, and R₂ and R₃ areindependently selected from C₁-C₁₀ alkyl, or alternatively R₂ and R₃,together with the nitrogen to which they are attached, form a 5- or6-membered saturated ring which may optionally contain between 1 and 3additional nitrogen atoms, and which may optionally be substituted withan aryl or heteroaryl group.

In a further embodiment T is CH₂, R₁ is Y-aryl or Y-heteroaryl where Yis a bivalent hydrocarbon having 1 to 4 carbon atoms, and the aryl groupis selected from: phenyl, biphenyl, naphthyl, anthracenyl andphenanthrenyl, and the heteroaryl group is a 5- or 6-membered ringwherein between 1 and 4 carbon atoms are replaced with nitrogen and/orsulfur atoms, and R₂ and R₃ are independently selected from C₁-C₆ alkyl,or alternatively R₂ and R₃, together with the nitrogen to which they areattached, form a saturated 5- or 6-membered ring which may optionallycontain 1 or 2 additional nitrogen atoms, and which may optionally besubstituted with a substituent selected from the group consisting of:pyrimidinyl, naphthyl, phenyl, pyrazinyl, triazinyl, triazolyl,imidazolyl, tetrazolyl and pyrrolyl.

In a further embodiment T is CH₂, R₁ is Y-aryl or Y-heteroaryl where Yis a bivalent hydrocarbon having 1 to 3 carbon atoms, and the aryl groupis selected from: phenyl, biphenyl and naphthyl, and the heteroarylgroup is selected from thienyl, tetrazolyl, imidazolyl, triazolyl,thiazolyl, isothiazolyl and pyrrolyl, and R₂ and R₃ are independentlyselected from C₁-C₆ alkyl, or alternatively R₂ and R₃, together with thenitrogen to which they are attached, form a saturated 5- or 6-memberedring which optionally contains 1 additional nitrogen atom, and which mayoptionally be substituted with a substituent selected from the groupconsisting of: pyrimidinyl, naphthyl, phenyl, pyrazinyl, triazinyl,triazolyl, imidazolyl, tetrazolyl and pyrrolyl.

In a further embodiment T is CH₂, R₁ is Y-aryl or Y-heteroaryl where Yis a bivalent hydrocarbon having 1 or 2 carbon atoms, and the aryl groupis selected from: phenyl, biphenyl and naphthyl, and the heteroarylgroup is selected from thienyl, tetrazolyl, imidazolyl, triazolyl,thiazolyl, isothiazolyl and pyrrolyl, and R₂ and R₃ are independentlyselected from C₁-C₆ alkyl, or alternatively R₂ and R₃, together with thenitrogen to which they are attached, form a saturated 5- or 6-memberedring which optionally contains 1 additional nitrogen atom, and which mayoptionally be substituted with a substituent selected from the groupconsisting of: pyrimidinyl, phenyl, pyrazinyl and triazinyl.

In a further embodiment T is CH₂, R₁ is Y-aryl or Y-heteroaryl where Yis —CH₂—, and the aryl group is selected from: phenyl, biphenyl andnaphthyl, and the heteroaryl group is selected from thienyl, tetrazolyl,imidazolyl, triazolyl, thiazolyl, isothiazolyl and pyrrolyl and R₂ andR₃ are independently selected from C₁-C₆ alkyl, or alternatively R₂ andR₃, together with the nitrogen to which they are attached, form asaturated 5- or 6-membered ring which optionally contains 1 additionalnitrogen atom, and which may optionally be substituted with asubstituent selected from the group consisting of: pyrimidinyl, phenyl,pyrazinyl and triazinyl.

In a further embodiment T is CH₂, R₁ is Y-aryl or Y-heteroaryl where Yis —CH₂—, and the aryl group is selected from: phenyl, biphenyl andnaphthyl, and the heteroaryl group is selected from thienyl, tetrazolyl,imidazolyl, triazolyl, thiazolyl, isothiazolyl and pyrrolyl, and R₂ andR₃ are independently selected from methyl, ethyl and propyl, oralternatively R₂ and R₃, together with the nitrogen to which they areattached, form a saturated 5- or 6-membered ring which optionallycontains 1 additional nitrogen atom, and which may optionally besubstituted on the additional nitrogen with a substituent selected fromthe group consisting of: pyrimidinyl, naphthyl, phenyl, pyrazinyl andtriazinyl.

In a further embodiment T is CH₂, R₁ is Y-aryl or Y-heteroaryl where Yis —CH₂—, and the aryl group is selected from: phenyl, biphenyl andnaphthyl, and the heteroaryl group is selected from thienyl, tetrazolyl,imidazolyl, triazolyl and pyrrolyl, and R₂ and R₃ are independentlyselected from C₁-C₆ alkyl, or alternatively R₂ and R₃, together with thenitrogen to which they are attached, form a saturated 5- or 6-memberedring which optionally contains 1 additional nitrogen atom, and which mayoptionally be substituted on the additional nitrogen with a substituentselected from the group consisting of: pyrimidinyl, phenyl, pyrazinyland triazinyl.

In a further embodiment T is CH₂, R₁ is Y-aryl or Y-heteroaryl where Yis —CH₂—, and the aryl group is selected from: phenyl and naphthyl, andthe heteroaryl group is selected from thienyl, tetrazolyl, imidazolyland triazolyl, and R₂ and R₃ are independently selected from C₁-C₆alkyl, or alternatively R₂ and R₃, together with the nitrogen to whichthey are attached, form a saturated 5- or 6-membered ring whichoptionally contains 1 additional nitrogen atom, and which may optionallybe substituted on the additional nitrogen with a substituent selectedfrom the group consisting of: pyrimidinyl and phenyl.

In one embodiment T is CH₂, R₁ is selected from the group consisting of:—CH₂-phenyl, —CH₂-thienyl and —CH₂-tetrazolyl, and R₂ and R₃ areindependently selected from C₁-C₆ alkyl, or alternatively R₂ and R₃,together with the nitrogen to which they are attached, form a structureselected from the group consisting of:

In still a further embodiment T is CH₂, R₁ is selected from the groupconsisting of: —CH₂-phenyl, —CH₂-thienyl and —CH₂-tetrazolyl, and R₂ andR₃ are independently selected from methyl, ethyl and propyl, oralternatively R₂ and R₃, together with the nitrogen to which they areattached, form a structure selected from the group consisting of:

The compound of the formula (I) may have the following structure:

wherein R₁, R₂, R₃ and T are as defined above and herein.

When exposed to a β-lactamase or transpeptidase the compounds of theformula (I) ultimately undergo an elimination reaction which results inthe liberation of nitric oxide.

Accordingly, the compounds of formula (I) represent nitric oxideprodrugs. The mechanism by which nitric oxide is liberated from thecompounds of the formula (I) is shown below in Scheme 1. Because theelimination reaction is instigated by exposure to a β-lactamase ortranspeptidase, an enzyme specific to bacteria, the release of nitricoxide from the compounds of formula (I) can be localised in the vicinityof a biofilm thereby minimising side effects and toxicity at otherlocations that may be associated with the uncontrolled release of nitricoxide.

The rate of release of nitric oxide from the compounds of the formula(I) may be modulated by altering the R₂ and/or R₃ substituents. Releasetimes in the order of a few seconds may be achieved when R₂ and R₃,together with the nitrogen to which they are attached, form apyrrolidinyl ring, whereas release times in the order of a few minutesare possible when R₂ and R₃ are lower alkyl groups, such as ethyl forexample. Release times in the order of about 3 to 20 hours may beachieved when R₂ and R₃ are lower alkyl groups having terminal aminosubstituents. Accordingly, an appropriate release rate of nitric oxidefrom the compounds of the formula (I) dependent on the intendedapplication is achievable by selecting appropriate R₂ and R₃substituents. Accordingly, those skilled in the art will recognise thatR₂ and R₃ may be selected from amongst a broad range of organic residuesdepending on the desired release time.

Because of its remoteness from the β-lactam ring and diazeniumdiolate,the substituent R₁ exerts minimal effect on the reaction of aβ-lactamase with the compounds of the formula (I) and the subsequentelimination of nitric oxide. Accordingly, those skilled in the art willrecognise that R₁ is not limited to the specific substituents definedherein, but rather may represent any organic residue.

In particular embodiments, the compounds of the disclosure comprise acephalosporin core or nucleus linked to a diazeniumdiolate. In furtherparticular embodiments the cephalosporin is cephaloram. One exemplarycompound of formula I provided by the present disclosure has thefollowing structure. Both the free carboxylic acid and carboxylate salts(e.g. K⁺ salt) are contemplated

In additional embodiments the R₂ and/or R₃ substituents may furthercomprise an antibiotic which is liberated from the compounds of theformula (I) together with nitric oxide. The concomitant release ofnitric oxide and the antibiotic may act in concert to more effectivelykill biofilm microorganisms; the nitric oxide inducing and promoting thedispersal of microorganisms from a biofilm and the antibiotic acting onthe dispersing cells. Those skilled in the art will appreciate that anysuitable antibiotic may be linked to the R₂ and/or R₃ substituents ofcompounds defined herein, with the selection of the appropriateantibiotic depending on factors such as the identity of thebiofilm-forming microorganisms, the extent of the biofilm and theenvironment in which the biofilm is located. The antibiotic may be anantibiotic comprising an NH group to facilitate formation of anantibiotic diazeniumdiolate conjugate. In one embodiment the antibioticis ciprofloxacin or a related antibiotic such as N-desmethyllevofloxacin.

The compounds of the present disclosure may be prepared by coupling the—O—N═N⁺(O)—N(R₂(R₃) moiety to X, either via a linker or a direct bond.In one embodiment, exemplary compounds of the formula (I) may beprepared according to Scheme 2.

Compounds of the formula (I) wherein the R₂ and/or R₃ substituentsfurther comprise an antibiotic may be prepared by the method depicted inScheme 3.

Whilst the antibiotic depicted in the method is ciprofloxacin thoseskilled in the art will appreciate that the method is applicable toother antibiotics that can be converted to diazeniumdiolate derivatives.

Typically, biofilms to be treated in accordance with embodimentsdescribed herein include microorganisms that express, or can be inducedto express an enzyme such as a β-lactamase or a transpeptidase.Induction of expression of a β-lactamase may be achieved by pretreatmentof microorganisms or biofilms with a suitable β-lactam antibiotic, oradministration of a suitable β-lactam antibiotic together withadministration of the compounds of formula (I). The β-lactam antibioticmay induce production of extracellular β-lactamase in saidbiofilm-forming microorganisms. The β-lactam antibiotic may beadministered at any suitable concentration, which may be subinhibitory,bacteriostatic or bacteriocidal. The β-lactamase is one that is capableof recognising and cleaving a β-lactam ring. Suitable β-lactamantibiotics for use in inducing β-lactamase expression where necessarymay therefore vary depending on the microorganisms to which thecompounds of the formula (I) are to be delivered. Those of ordinaryskill in the art would readily be able to determine the appropriateβ-lactam antibiotic to be employed.

Accordingly, compounds, compositions and methods of the presentdisclosure enable the spatial and temporal control of nitric oxiderelease thereby facilitating the targeted delivery or administration ofa nitric oxide generator or releasing agent to a desired site comprisinga biofilm or at which a biofilm may form, and the release of nitricoxide with appropriate kinetics for the particular application.

Those skilled in the art will appreciate that embodiments of the presentdisclosure are applicable to single species or mixed species biofilms.Bacterial species to which the present invention relates may be anyspecies capable of forming a biofilm or contributing to a biofilm andwhich produce or can be induced to produce a β-lactamase. Species mayinclude, but are not limited to, Pseudomonas spp. such as P. aeruginosa,Pseudoalteromonas spp. such as P. tunicata, Staphylococcus spp. such asS. aureus and S. epidermidis, Streptococcus spp., Escherichia spp. suchas E. coli, Shigella spp., Mycobacterium spp., Enterococcus spp.,Salmonella spp., Legionella spp., Haemophilus spp., Bacillus spp.,Desulfovibrio spp., Shewanella spp., Geobacter spp., Klebsiella spp.such as K. pneumoniae, Proteus spp. such as P. mirabilis, Serratia spp.such as S. marcescens, Porhyromonas spp., Fusobacterium spp., Proteusspp., Aeromonas spp., Arthrobacter spp., Micrococcus spp., andBurkholderia spp. Alternatively those skilled in the art will appreciatethat in some applications of the present invention, the identities ofthe particular species in the mixed communities of the biofilm to betreated are undetermined and are not critical to the applicability ofthe invention.

In accordance with embodiments disclosed herein, compounds of theformula (I) are typically used in amounts such that a low, non-toxicconcentration of nitric oxide is released in the vicinity of the biofilmor biofilm-forming microorganisms. The concentration may be in thenanomolar, micromolar, or millimolar range. In particular embodiments,the concentration may be between about 1 nM and about 100 mM, betweenabout 10 nM and about 50 mM, between about 25 nM and about 50 mM,between about 50 nM and about 25 mM, between about 100 nM and about 10mM, between about 200 nM and about 1 mM, between about 500 nM and 500μM, between about 500 nM and 100 μM, or between about 1 μM and about 50μM. The most suitable concentration to achieve the desired effect willdepend on a number of factors and may be determined by those skilled inthe art using routine experimentation. Such factors include, but are notlimited to, the particular compound used for nitric oxide release, themeans or route of administration of the compound, the nature, structureand age of the biofilm, the species of microorganism to be treated andso on.

Compounds, compositions and methods of the present disclosure may beemployed in combination with at least one additional antibiotic orantimicrobial agent. As hereinbefore described the compounds of thepresent disclosure may incorporate an antibiotic linked to the R₂ and/orR₃ substituents. Alternatively, or in addition, compounds of the presentdisclosure may be administered or delivered in conjunction with one ormore antibiotics or antimicrobial agents, either simultaneously orsequentially. For sequential application the antibiotics orantimicrobial agents may be formulated into the same composition as thecompounds of the present disclosure. By way of example only, suitableantibiotics include but are not limited to β-lactams, monopenems,carboxypenems, aminoglycosides, quinolones, macrolides, lincozamides,tetracyclines, streptogramins, glycopeptides, rifamicins, sulphonamideschloramphenicol, nalidixic acid, azole-containing compounds and peptideantibiotics. Exemplary antibiotics include ceftazidime and tetracycline.

Suitable antimicrobial agents include, but are not limited to,detergents, surfactants, agents that induce oxidative stress,bacteriocins and antimicrobial enzymes, peptides and phage.Antimicrobial enzymes include but are not limited to lipases, pronases,lyases (e.g. alginate lyases) and various other proteolytic enzymes andnucleases. The antibiotics and antimicrobial agents may be natural orsynthetic. The antibiotic or antimicrobial agent employed may beselected for the particular application of the invention on acase-by-case basis, and those skilled in the art will appreciate thatthe scope of the present invention is not limited by the nature oridentity of the particular antimicrobial agent.

The compounds, compositions and methods disclosed herein findapplication in a wide range of environments and circumstances. Thefollowing is a brief discussion of some general areas of application.However those skilled in the art will readily appreciate that anyenvironment or situation in which biofilm development is a problem or inwhich it is desirable to inhibit microbial growth will be potentiallysuitable.

Compounds, compositions and methods of the present disclosure findparticular application in the treatment, prevention and ongoingmanagement of infectious diseases and of diseases and disordersassociated with, characterised by, or caused by biofilms andbiofilm-forming microorganisms. For example, a variety of bacterialinfections associated with biofilm formation may be treated with methodsand compositions of the invention, such as cystic fibrosis, otitismedia, bacterial endocarditis, kidney stones, legionnaire's disease,urinary tract infections, pulmonary infections, dental plaque, dentalcaries and infections associated with surgical procedures or burns.Accordingly, compositions of the invention may be formulated aspharmaceutical compositions or form components of, for example, surgicaldressings, mouthwash, toothpaste or saline solutions.

Compounds and compositions of the present disclosure may be included inpharmaceutical, cosmetic, dermatological or topical deliverycompositions as preservatives to inhibit or prevent the growth and/orcolonisation of unwanted microorganisms. The compositions of theinvention are therefore useful for preventing spoilage and henceincreasing the usable life of any type of pharmaceutical, cosmetic,dermatological or topical delivery compositions to which they are added.The compounds or compositions of the disclosure may be convenientlyincluded in any solid or liquid pharmaceutical, cosmetic, dermatologicalor topical delivery composition during the manufacture thereof, oralternatively after manufacture. The term “cosmetic composition” isunderstood to mean a composition intended for placement in contact withany external part of an animal body, including the mucous membranes ofthe oral cavity, the teeth, the hair and the nails, for the purpose of,for example: protecting, perfuming, cleansing, maintaining (i.e.moisturising or exfoliating), beautifying, altering the appearance of,or altering the odour of, the body. Examples of cosmetic compositionsinclude but are not limited to: nail care products, make up, productsintended for application to the lips, face masks and scrubs, hair tints,dyes and bleaches, products for waving, straightening and fixing hair,cleansing products such as lotions, powders and shampoos, conditioningproducts such as. lotions, creams, oils, hairdressing products such aslotions and lacquers, products for care of the teeth and the mouth,including toothpastes, mouthwashes, tongue cleaners, dentalbleaches/whiteners and denture cleansers, perfumes, toilet waters, Eaude colognes, feminine hygiene products, deodorants, antiperspirants,cleansers such as toilet soap, deodorant soap, astringent and skinwashes, shaving products such as creams, foams and lotions, bath andshower preparations such as salts, foams, oils, gels, etc.,depilatories, after-bath powders, hygienic powders, moisturisingproducts such as creams, lotions, gels and foams, sunbathing products(without SPF or SPF<4), anti-wrinkle products (without SPF) andanti-ageing products (without SPF).

Compounds, compositions and methods of the present disclosure may alsobe used in coating medical devices, including medical and surgicalequipment and implantable medical devices, including but not limited tovenous catheters, drainage catheters (e.g. urinary catheters), stents,pacemakers, contact lenses, hearing-aids, percutaneous glucose sensors,dialysis equipment, drug-pump related delivery cannula, prostheses suchas artificial joints, hearts, heart valves or other organs, medicalfixation devices (e.g. rods, screws, pins, plates and the like).Further, embodiments of the present disclosure find application in woundrepair, as for example, compounds and compositions comprising the samemay be impregnated or coated onto sutures and wound dressings such asbandages.

Compounds, compositions and methods of the present disclosure also findapplication in a range of industrial and domestic applications,including but not limited to water supply reservoirs and feed pipes,drain pipes (domestic or industrial scale), process equipment of, forexample, cooling towers, water treatment plants, dairy processingplants, food processing plants, chemical manufacturing plants,pharmaceutical or biopharmaceutical manufacturing plants, oil pipelinesand oil refinery equipment, and pulp and paper mills. Other amenableenvironments and settings include, for example, as marine anti-foulingpaints or coatings, for example in treating ship hulls, aquacultureequipment, fishing nets or other in-water structures.

Compositions according to the present disclosure may be in any suitableform. Typically the form will depend on that which is most suitable forapplication or delivery to the required site and thus will vary withdifferent medical, industrial and domestic applications. For example acomposition may be formulated for in vivo administration, such as in theform of a liquid, suspension, nasal spray, eyedrops, powder, tablet,capsule, cream, paste, gel or lotion. For industrial and domesticapplications the composition may be formulated as a paint, wax, othercoating, emulsion, solution, gel, suspension, beads, powder, granules,pellets, flakes or spray. The skilled addressee will also recognise thatthe appropriate formulation will depend on the particular applicationand the proposed route of delivery. Suitable routes of administrationfor in vivo applications include, for example, oral, nasal, parenteral(e.g. intravenous, topical, intraarterial, intramuscular, intraocular),transdermal and subcutaneous administration.

Compositions of the invention typically also include carriers, diluentsor excipients. Suitable carriers, diluents and excipients are known tothose skilled in the art. The diluents, adjuvants and excipients must be“acceptable” in terms of being compatible with the other ingredients ofthe composition, and in the case of pharmaceutical compositions, notdeleterious to the recipient thereof. Carriers may be liquid or solid.In the case of liquid carriers, the liquid may be an aqueous ornon-aqueous solvent.

In addition to the controlled release of nitric oxide provided by thecompounds of the present disclosure per se, a further level ofcontrolled release may be desirable and may be imparted by theformulation of compounds into compositions. For pharmaceuticalapplications, a number of suitable controlled release systems are knownin the art. For example, polymeric colloidal particles ormicroencapsulates (microparticles, microspheres or nanoparticles) in theform of reservoir and matrix devices may be employed, or the agent maybe contained by a polymer containing a hydrophilic and/or leachableadditive eg, a second polymer, surfactant or plasticiser, etc. to give aporous device, or a device in which the drug release may be osmotically‘controlled’ (both reservoir and matrix devices).

Large cage-like molecules such as the C₆₀ Buckminster-fullerenes(‘Buckyballs’) or hyperbranched (starburst) dendrimers may also be used.

Typically for anti-fouling and other industrial applications, thecomposition, for example in the form of a paint or other surfacecoating, employs a carrier enabling the controlled release of the activeagent temporally and/or spatially. A variety of methods to achievecontrolled release of bioactive agents are known to those skilled in theart and may include, for example, encapsulation of the active agent in asuitable polymer or polymer-based product. The polymer may be an organicor inorganic polymer, for example a polyolefin, polyether, polyester,polyamide, polyurethane or polypeptide. Suitable polymers for providingcontrolled release are known to those skilled in the art, for example asdisclosed in U.S. Pat. No. 6,610,282, the disclosure of which isincorporated herein by reference.

Typically, the rate of release of the substance is determined by theproperties of the polymer itself as well as environmental factors (suchas pH, temperature etc). Controlled release systems are capable ofdelivering substances slowly and continuously for up to several years.Those skilled in art will appreciate that a number of controlled releasesystems are applicable to the delivery of agents according to thepresent invention. By way of example only, release may be diffusioncontrolled, chemically controlled or solvent activated.

In diffusion controlled systems, diffusion of the agent trapped within apolymer matrix is the rate-determining factor for the overall releaserate. One type of diffusion controlled system employs a reservoir devicein which the agent forms a core surrounded by an inert diffusionbarrier. These systems include membranes, capsules, microcapsules,liposomes, and hollow fibers. Alternatively the device may be amonolithic device in which the active agent is dispersed or dissolved inan inert polymer. Diffusion through the polymer matrix is therate-limiting step, and release rates are determined in part by thechoice of polymer and its consequent effect on the diffusion andpartition coefficient of the agent to be released.

In typical chemically controlled systems a polymer degrades over timeand releases an agent in an amount proportional to the gradual erosion.Chemical control can be achieved using bioerodible or pendant chains. Ina bioerodible system the agent is ideally distributed uniformlythroughout a polymer in the same way as in monolithic diffusion systems.As the polymer surrounding the agent is eroded, the agent escapes. In apendant chain system, the agent is covalently bound to the polymer andis released by bond scission owing to water or enzymes.

In typical solvent-activated controlled systems, the active agent isdissolved or dispersed within a polymeric matrix and is not able todiffuse through that matrix. Osmotic pressure is used as the drivingforce for release of the agent. In one type of solvent-controlledsystem, as the environmental fluid (e.g., water) penetrates the matrix,the polymer (e.g a hydrogel) swells and its glass transition temperatureis lowered below the environmental (host) temperature. Thus, the swollenpolymer is in a rubbery state and allows the drug contained within todiffuse through the encapsulant.

Chemical bonding of a bioactive agent to a polymer can be accomplishedin several general ways based on different methods of synthesis wellknown to those skilled in the art including: reaction on preformedpolymers; reactions on naturally-occurring polymers; polymerization ofvinyl monomers containing the active ingredient; and step growthpolymerizations. When the bioactive agent is chemically bonded to apolymer, the bond has to be cleaved by a chemical reaction—typicallyenzymatic, hydrolytic, thermal, or photochemical. A variety of chemicaland physical variables can affect the rate of bond cleavage andsubsequent release of chemically attached materials from polymersincluding the nature of the labile bone, length of the spacer group,molecular weight, hydrophilicity, neighbouring group effects,environmental factors and physical form and dimensions.

In anti-fouling applications, self-polishing antifouling coatings areknown in the art. Such coatings are typically based on polymers oftributyltin methacrylate, methyl methacrylate, and film softeningmonomers such as 2-ethylhexyl acrylate. An organotin polymer typicallyacts as the paint binder. Such paints may also contain a toxicantadditive such as cuprous oxide or a triorganotin compound. In addition,the usual paint additives such as pigments, thixotropic agents may alsobe present. In normally alkaline seawater, the polymeric organotinbinder is gradually hydrolyzed, and the tributyltin is liberated in aform that is an active antifoulant. The hydrolyzed polymer formed iswater-soluble or water-swellable and is easily eroded off the surface bymoving seawater, exposing a fresh surface of paint.

Those skilled in the art will readily appreciate that the deliverysystems and methods described above are merely examples of suitablemethods and systems that may be employed in the present invention. Anyother suitable carriers and delivery systems may be employed to achievethe desired means of application of agents according to embodiments ofthe present invention.

Examples of pharmaceutically acceptable diluents are demineralised ordistilled water; saline solution; vegetable based oils such as peanutoil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oilssuch as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil,sesame oil, arachis oil or coconut oil; silicone oils, includingpolysiloxanes, such as methyl polysiloxane, phenyl polysiloxane andmethylphenyl polysolpoxane; volatile silicones; mineral oils such asliquid paraffin, soft paraffin or squalane; cellulose derivatives suchas methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols,for example ethanol or iso-propanol; lower aralkanols; lowerpolyalkylene glycols or lower alkylene glycols, for example polyethyleneglycol, polypropylene glycol, ethylene glycol, propylene glycol,1,3-butylene glycol or glycerin; fatty acid esters such as isopropylpalmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone;agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly.Typically, the carrier or carriers will form from 1% to 99.9% by weightof the compositions.

For pharmaceutical applications, compositions may be formulated fordelivery by any route, for example oral, topical, intracavitary,intravesical, intramuscular, intraarterial, intravenous or subcutaneous.

Those skilled in the art will appreciate that the aspects andembodiments described herein are susceptible to variations andmodifications other than those specifically described. It is to beunderstood that the disclosure includes all such variations andmodifications. The disclosure also includes all of the steps, features,compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations of any two or more of said steps or features.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the presentapplication. Further, the reference in this specification to any priorpublication (or information derived from it), or to any matter which isknown, is not, and should not be taken as an acknowledgment or admissionor any form of suggestion that that prior publication (or informationderived from it) or known matter forms part of the common generalknowledge in the field of endeavour to which this specification relates.

The present disclosure is further described by reference to thefollowing non-limiting examples.

EXAMPLES Example 1 Synthesis of Compounds

The following representative compounds of the formula (I) weresynthesised:

The compounds were synthesised according to Scheme 4:

The first step in the synthesis of compounds 14 to 25 involvedpreparation of cephalosporin-3′-diazeniumdiolates 3 and 9-13 by reactionof appropriately functionalised PMB-protected cephalosporins 1, 4 and 5with appropriately functionalised diazeniumdiolates 2 and 6 to 8, asshown below:

Starting Sodium Pro- Yield material, R¹ Diazeniumdiolate, Z duct %^(a) 1

2 NEt₂ 3 85 4

2 NEt₂ 9 75^(b) 5

2 NEt₂ 10 14^(c) 4

6

11 66 4

7

12 39 4

8

13 80 ^(a)Isolated yield of pure Δ2-isomer, ^(b)Δ3-isomer typicallyisolated in ~5% yield, ^(c)42% of pure Δ3-isomer isolated.

Compound 3 was prepared utilising the following method.

Sodium iodide (0.912 g, 6.08 mmol) was added to a suspension of thePMB-protected cephalosporin ester 1 (3.00 g, 6.08 mmol) in anhydrousacetone (25 mL) under N₂ and the mixture was stirred in the dark at roomtemperature for 1 h. Sodium(Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate 2 (0.944 g, 6.08 mmol)was then added in one shot and the mixture stirred at room temperaturefor a further 1.5 hrs (TLC analysis; Pet Spirit:EtOAc 7:3). The solventwas removed under reduced pressure and the residue diluted with CH₂Cl₂(75 mL) and washed with 10% aq. sodium thiosulphate (2×40 mL) and water(1×40 mL). The organic fraction was dried over anhydrous MgSO₄ andconcentrated in vacuo. The crude product was purified by silica gelcolumn chromatography (Pet. Spirit:EtOAc, 7:3) and recrystallised fromEtOH or MeOH to give 3 (3.04 g, 85%) as a pale yellow powder. ¹H NMR(500 MHz, CDCl₃): δ 7.32 (d, 2H, J=8.5 Hz), 7.26 (d, 1H, J=5 Hz),7.00-6.96 (m, 2H), 6.89 (d, 2H, J=8.5 Hz), 6.34 (d, 1H, J=9 Hz), 5.81(dd, 1H, J=10, 4.5 Hz), 5.34 and 4.99 (ABq, 2H, J=14.5 Hz), 5.18 (s,2H), 4.90 (d, 1H, J=5 Hz), 3.84 (s, 2H), 3.80 (s, 3H), 3.52 and 3.44(ABq, 2H, J=18.5 Hz), 3.11 (q, 4H, J=7.5 Hz), 1.05 (t, 6H, J=7.5 Hz).¹³C NMR (500 MHz, CDCl₃): δ 169.8, 164.4, 161.2, 159.9, 134.6, 130.6,127.8, 127.5, 126.6, 126.4, 126.0, 125.4, 113.9, 71.9, 68.1, 59.2, 57.4,55.2, 48.3, 37.1, 25.9 11.5. FTIR (cm⁻¹, Neat): 3275, 1754, 1706, 1648,1517, 1362, 1248, 1177, 1096, 1027, 822. M.P 166° C. [α]_(D) (c=1.0,CHCl₃)=+39.0. ESI-HRMS (m/z) Calcd. for 588.1592 [M−H]⁻ C₂₆H₃₀N₅O₇S₂ ⁻;Found 588.1550.

Compounds 9 to 13 were prepared using this same method by selectingappropriately functionalised PMB-protected cephalosporins anddiazeniumdiolates. Spectroscopic data for compounds 9 to 13 is presentedbelow.

Compound 9

¹H NMR (500 MHz, CDCl₃): δ 7.36-7.24 (m, 7H), 6.88 (d, 2H, J=9 Hz), 6.08(d, 1H, J=10 Hz), 5.81 (dd, 1H, J=10, 4.5 Hz), 5.33 and 4.98 (ABq, 2H,J=14 Hz), 5.17 (s, 2H), 4.88 (d, 1H, J=5 Hz), 3.79 (s, 3H), 3.67 and3.62 (ABq, 2H, J=9 Hz), 3.44 and 3.42 (ABq, 2H, J=18 Hz), 3.10 (q, 4H,J=7 Hz), 1.05 (m, 6H, J=7 Hz). ¹³C NMR (500 MHz, CDCl₃): 171.1, 164.6,161.2, 159.9, 133.6, 130.7, 129.4, 129.2, 127.8, 126.7, 126.4, 125.5,114.0, 72.0, 68.1, 59.2, 57.5, 55.2, 48.4, 43.3, 26.0, 11.5. FTIR (cm⁻¹,Neat): 3284, 1778, 1726, 1660, 1519, 1352, 1228, 1187, 1030, 982, 818,716, 699, 679. M.P 126-128° C., [α]_(D) (c=1.0, CH₂Cl₂)=+76.9, ESI-HRMS(m/z) Calcd. for 584.2179 [M+]⁺ C₂₈H₃₄N₅O₇S, Found 584.2205.

Compound 10

¹H NMR (500 MHz, CD₃OCD₃): δ 8.90 (s, 1H), 8.26 (d, 1H, J=6 Hz), 7.30(d, 2H, J=8.5 Hz), 6.86 (d, 2H, J=8.5 Hz), 5.78 (q, 1H, J=5 Hz), 5.32and 5.27 (ABq, 2H, J=16.5 Hz), 5.18 (s, 2H), 5.17 and 5.02 (ABq, 2H,J=13 Hz), 4.95 (d, 1H, J=5 Hz), 3.78 (s, 3H), 3.56 and 3.51 (ABq, 2H,J=19 Hz), 3.14 (q, 4H, J=7 Hz), 1.05 (t, 6H, J=7 Hz). ¹³C NMR (125 MHz,CD₃OD): δ 166.3, 164.4, 161.5, 160.2, 144.5, 130.0, 126.8, 126.6, 126.3,114.2, 72.1, 68.5, 59.7, 57.5, 55.5, 50.1, 48.4, 26.5, 11.7. FTIR (cm⁻¹,Neat): 3290, 3137, 2973, 2902, 1771, 1702, 1662, 1556, 1378, 1233, 1170,1094, 1049, 801. M.P 171° C., [α]_(D) (c=1.0, acetone)=−51.9, ESI-HRMS(m/z) Calcd. for 574.1838 [M−H]⁻ C₂₃H₂₈N₉O₇S⁻, Found 574.1830.

Compound 11

¹H NMR (500 MHz, CDCl₃): δ 7.36 (m, 2H), 7.32 (d, 2H, J=8.5 Hz), 7.26(m, 3H), 6.88 (d, 1H, J=8.5 Hz), 6.05 (d, 1H, J=9 Hz), 5.82 and 5.80(dd, 1H, J=4.5, 5 Hz), 5.21-5.15 (m, 3H), 4.91 (d, 1H, J=4.4 Hz), 4.87(d, 1H, J=13.5 Hz), 3.80 (s, 3H), 3.68 and 3.62 (ABq, 2H, J=16 Hz), 3.52and 3.43 (ABq, 2H, J=18.5 Hz), 3.46 (t, 4H, J=7 Hz), 1.91 (t, 4H, J=7Hz). ¹³C NMR (125 MHz, CDCl₃): 171.0, 164.7, 161.3, 159.9, 133.5, 130.6,129.4, 129.2, 127.8, 126.8, 126.7, 125.3, 114.0, 71.4, 68.0, 59.1, 57.4,55.2, 50.7, 43.3, 26.1, 22.8. FTIR (cm⁻¹, Neat): 3265, 2965, 2162, 2030,1756, 1714, 1652, 1612, 1536, 1486, 1446, 1392, 1266, 1244, 1217, 1180,1013, 986. M.P 157° C. [α]_(D) (c=1.0, MeOH) =+20.6. ESI-HRMS (m/z)Calcd. for 580.1871 [M−H]⁻ C₂₈H₃₀N₅O₇S⁻¹; Found 580.1895.

Compound 12

¹H NMR (500 MHz, CDCl₃): δ 8.33 (d, 2H, J=4.5 Hz), 7.39-7.26 (m, 5H),7.32 (d, 2H, J=7.5 Hz), 6.88 (d, 2H, J=7.5 Hz), 6.56 (t, 1H, J=2 Hz),6.18 (d, 1H, J=9 Hz), 5.82 (q, 1H, J=5 Hz), 5.24 and 4.95 (ABq, 2H,J=13.5 Hz), 5.18 (d, 2H, J=2.5 Hz), 4.91 (d, 1H, J=4.5 Hz), 3.98 (t, 4H,J=4.5 Hz), 3.78 (s, 3H), 3.66 and 3.61 (ABq, 2H, J=16 Hz), 3.53 and 3.40(ABq, 2H, J=18.5 Hz), 3.41 (t, 4H, J=4.5 Hz). ¹³C NMR (500 MHz, CDCl₃):δ 171.0, 164.7, 161.3, 159.9, 157.8, 133.6, 130.7, 129.4, 129.2, 127.7,126.6, 125.8, 125.7, 113.9, 110.7, 71.8, 68.1, 59.2, 57.4, 55.2, 50.9,43.3, 42.3, 26.1. FTIR (cm⁻¹, Neat): 3286, 3137, 2976, 2908, 2904, 1772,1702, 1662, 1557, 1411, 1377, 1232, 1049. [α]_(D) (c=1.0, MeOH)=+39.5.ESI-HRMS (m/z) Calcd. for 675.2344 [M+H]⁺ C₃₂H₃₅N₈O₇S⁺, Found 675.2373.M.P 136° C.

Compound 13

¹H NMR (500 MHz, CDCl₃): δ 7.36-7.24 (m, 6H), 6.93-6.91 (m, 2H), 6.88(d, 2H, J=9 Hz), 6.16 (m, 1H), 5.81 (q, 1H, J=5 Hz), 5.24 (d, 1H, J=2.5Hz), 5.21 (s, 2H), 5.17 and 4.96 (ABq, 2H, J=12 Hz), 4.91 (d, 2H, J=2.5Hz), 3.78 (s, 3H), 3.67 and 3.58 (ABq, 2H, J=16 Hz), 3.53 and 3.40 (ABq,2H, J=18.5 Hz), 3.51 (m, 4H), 3.29 (m, 4H). ¹³C NMR (125 MHz, CDCl₃): δ171.3, 164.9, 161.5, 160.2, 150.4, 133.8, 130.9, 129.6, 129.5, 129.4,127.9, 126.9, 126.0, 121.0, 116.8, 114.2, 72.0, 68.4, 59.4, 57.7, 55.4,51.1, 48.4, 43.5, 26.1. FTIR (cm⁻¹, Neat): 3389, 3286, 3197, 3030, 2897,1756, 1647, 1607, 1546, 1492, 1448, 1384, 1351, 1224, 1004. [α]_(D)(c=1.0, MeOH)=+54.3, ESI-HRMS (m/z) Calcd. for 673.2439 [M+H]⁺C₃₄H₃₇N₆O₇S⁺, Found 673.2409. M.P 106° C.

The PMB-protected cephalosporin-3′-diazeniumdiolates (3 and 9 to 13)were then deprotected using neat trifluoroacetic acid to give the freecarboxylic acids.

R⁶ = R⁶ = OH O^(⊖)K^(⊕) Es- Yield Yield ter R¹ Z % % 3

NEt₂ 14, 36 20, 99 9

NEt₂ 15, 80 21, 99 10

NEt₂ 16, 81 22 11

17, 83 23, 98 12

18, 93 24, 94 13

19, 67 25

Compound 14 was prepared utilising the following method.

The PMB-protected cephalosporin-3′-diazeniumdiolate 3 (1.00 g, 1.69mmol) and anhydrous anisole (0.55 g, 5.09 mmol) were stirred at 0° C.for 30 mins with trifluoroacetic acid (4.0 mL) after which the mixturewas poured slowly onto crushed ice (˜100 g). Upon melting, the aqueousmixture was extracted with CH₂Cl₂ (3×70 mL) and the combined organicextracts were dried over anhydrous MgSO₄ and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(EtOAc:MeOH:H₂O, 85:15:0.5) to provide thecephalosporin-3′-diazeniumdiolate free acid 14 (0.285 g, 36%) as a paleyellow powder. ¹H NMR (500 MHz, CD₃OD): δ 7.26 (s, 1H, J=1 Hz),6.96-6.92 (m, 2H), 5.73 (d, 1H, J=5, Hz), 5.29 and 5.07 (ABq, 2H, J=12.9Hz), 5.05 (d, 1H, J=4.8 Hz), 3.83 and 3.79 (ABq, 2H, J=15 Hz), 3.62 and3.53 (ABq, 2H, J=18 Hz), 3.16 (q, 4H, J=7.0 Hz), 1.04 (t, 6H, J=7.0 Hz).¹³C NMR (500 MHz, CD₃OD): δ 173.3, 166.1, 164.5, 137.4, 130.2, 128.4,127.7, 126.9, 125.8, 73.0, 60.8, 59.0, 49.0, 37.1, 27.1, 11.7. FTIR(cm⁻¹, Neat): 3275, 2156, 1754, 1661, 1656, 1522, 1320, 1235, 1065, 995.M.P 87° C., [α]_(D) (c=1.0, MeOH)=+84.4, ESI-HRMS (m/z) Calcd. for468.1017 [M−H]⁻ C₁₈H₂₂N₅O₆S₂ ⁻, Found 468.0996.

Compounds 15 to 19 were prepared using this same method. Spectroscopicdata for compounds 15 to 19 is presented below.

Compound 15

¹H NMR (500 MHz, CD₃OD): δ 7.35-7.26 (m, 5H), 5.75 (d, 1H, J=4.8, Hz),5.36 and 5.07 (ABq, 2H, J=12.9 Hz), 5.05 (d, 1H, J=4.8 Hz), 5.03 (m,1H), 3.68-3.45 (m, 4H), 3.19 (q, 4H, J=7.0 Hz), 1.07 (t, 6H, J=7.0 Hz).¹³C NMR (500 MHz, CD₃OD): δ 175.4, 167.3, 166.6, 137.2, 132.1, 131.0,130.4, 128.8, 123.0, 74.6, 61.5, 59.8, 50.1, 44.0, 27.7, 12.6. FTIR(cm⁻¹, Neat): 3287, 1780, 1663, 1505, 1337, 1223, 998, 618. M.P 84-86°C., [α]_(D) (c=1.0, CH₂Cl₂)=+19.4, ESI-HRMS (m/z) Calcd. for 462.1453(C₂₀H₂₄N₅O₆S) [M−H]⁻, Found 462.1465.

Compound 16

¹H NMR (300 MHz, CD₃OCD₃): δ 9.40 (d, 1H, J=6 Hz), 9.11 (s, 1H), 5.72(d, 2H, J=8 Hz), 5.29 (s, 2H), 5.22 and 4.99 (ABq, 2H, J=22 Hz), 5.01(s, 1H), 3.52 and 3.49 (ABq, 2H, J=19 Hz), 3.10 (q, 4H, J=7 Hz), 0.95(t, 6H, J=7 Hz). ¹³C NMR (125 MHz, CD₃OD): δ 167.4, 165.8, 164.4, 146.0,128.1, 127.5, 72.9, 60.6, 58.7, 50.3, 49.3, 27.1, 11.7. FTIR (cm⁻¹,Neat): 3282, 3136, 2975, 2875, 1773, 1703, 1662, 1559, 1414, 1227, 1170,1026, 800. M.P 156° C. [α]_(D) (c=1.0, MeOH)=+117.3, ESI-HRMS (m/z)Calcd. for 454.1263 [M−H]⁻ C₁₅H₂₀N₉O₆S⁻, Found 454.1266.

Compound 17

¹H NMR (500 MHz, CD₃OD): δ 7.30-7.22 (m, 5H), 5.72 (d, 1H, J=7.5 Hz),5.23 and 4.97 (ABq, 2H, J=22.5 Hz), 5.07 (d, 1H, J=8 Hz), 3.64-3.50 (m,4H), 3.50 (t, 4H, J=7 Hz), 1.93 (t, 4H, J=7 Hz). ¹³C NMR (125 MHz,CDCl₃): 174.5, 166.2, 164.5, 136.4, 130.4, 129.5, 127.9, 127.6, 72.5,60.7, 59.1, 51.5, 43.1, 27.2, 23.7. FTIR (cm⁻¹, Neat): 3296, 1756, 1729,1642, 1530, 1469, 1429, 1388, 1318, 1141, 1028, 944. [α]_(D) (c=1.0,MeOH)=122.8, ESI-HRMS (m/z) Calcd. for 484.1261 [M+Na]⁺ C₂₀H₂₃N₅NaO₆S⁺,Found 484.1281. M.P 146° C.

Compound 18

¹H NMR (300 MHz, CD₃OCD₃): δ 8.37 (s, 2H), 7.34-7.23 (m, 5H), 6.65 (s,1H), 5.84 (s, 1H), 5.24 and 4.97 (ABq, 2H, J=7.8 Hz), 5.14 (s, 1H), 3.97(s, 4H), 3.69-3.41 (m, 4H), 3.46 (s, 4H). ¹³C NMR (75 MHz, CDCl₃):172.1, 166.4, 164.0, 163.0, 159.5, 137.5, 130.9, 129.9, 128.0, 126.7,111.9, 72.8, 61.5, 58.2, 52.0, 43.3, 42.3, 26.1. FTIR (cm⁻¹, Neat):3286, 3137, 2976, 2908, 2904, 1772, 1702, 1662, 1557, 1411, 1232, 1049.[α]_(D) (c=1.0, MeOH)=+36.6, ESI-HRMS (m/z) Calcd. for 555.1769 [M+H]⁺C₂₄H₂₇N₈O₆S⁺, Found 555.1799. M.P 116° C.

Compound 19

¹H NMR (500 MHz, CD₃COCD₃): δ 8.05 (d, 1H, J=8.5 Hz), 7.36-7.23 (m, 8H),7.02 (d, 2H, J=7.5 Hz), 6.84 (t, 1H, J=7.5), 5.85 (q, 1H, J=5 Hz), 5.25and 5.01 (ABq, 2H, J=13.5 Hz), 5.14 (d, 1H, J=4.5 Hz), 3.70 and 3.58 (m,4H), 3.55 (m, 4H), 3.35 (m, 4H). ¹³C NMR (500 MHz, CD₃COCD₃): 171.6,165.8, 163.1, 151.5, 136.5, 130.0, 129.8, 129.1, 127.4, 125.9, 120.7,117.2, 72.2, 60.3, 58.5, 51.6, 48.7, 42.9, 26.7. FTIR (cm⁻¹, Neat):3397, 3286, 3197, 3028, 2897, 1755, 1647, 1607, 1548, 1492, 1448, 1383,1351, 1225, 1004, 985. [α]_(D) (c=1.0, MeOH)=+57.7 ESI-HRMS (m/z) Calcd.for 553.1864 [M+H]⁺ C₂₆H₂₉N₆O₆S⁺, Found 553.1847. M.P 96° C.

Compounds 14, 15 17 and 18 were then converted to their potassium salts.Compound 20 was prepared utilising the following method.

Cephalosporin-3′-diazeniumdiolate free acid 14 (0.20 g, 0.42 mmol) wassuspended in H₂O (1.0 ml) at 0° C. to which was then added an ice-coldaqueous solution of KOH (0.023 g in 100 μL H₂O, 0.42 mmol). A paleyellow solution was rapidly formed and stirring was continued for afurther 20 minutes at 0° C. The aqueous solution was washed with CH₂Cl₂(2×2 mL) and then frozen and lyophilized to provide the potassiumcephalosporin-3′-diazeniumdiolate carboxylate salt 20 (0. 21 g, 99%) asa pale yellow powder. ¹H NMR (300 MHz, D₂O): δ 7.21 (d, 1H, J=10 Hz),6.89-6.88 (s, 2H), 5.49 (d, 1H, J=4.5 Hz), 5.08 and 4.85 (ABq, 2H,J=12.6 Hz), 4.96 (d, 1H, J=4.8 Hz), 3.81 and 3.74 (ABq, 2H, J=15 Hz),3.46 and 3.25 (ABq, 2H, J=18 Hz), 2.96 (q, 4H, J=7.0 Hz), 0.85 (t, 6H,J=7.0 Hz). ¹³C NMR (125 MHz, D₂O): δ 174.4, 168.3, 164.7, 135.8, 132.9,127.6, 127.5, 125.9, 115.4, 73.9, 59.4, 57.7, 49.0, 36.2, 25.5, 10.7.FTIR (cm⁻¹, Neat): 3395, 3326, 3210, 2883, 2816, 1600, 1369, 1233, 1034.[α]_(D) (c=1.0, MeOH)=+90.9, ESI-HRMS (m/z) Calcd. for 508.0721 [M+H]⁺C₁₈H₂₃N₅O₆S₂K⁺, Found 508.0742.

Compounds 21, 23 and 24 were prepared using this same method.Spectroscopic data for compounds 21, 23 and 24 is presented below.

Compound 21

¹H NMR (500 MHz, CD₃OD): δ 7.30-7.20 (m, 5H,), 5.65 (d, 1H, J=4.8, Hz),5.35 and 4.85 (ABq, 2H, J=12.0 Hz), 4.98 (d, 1H, J=4.8, Hz), 3.60 and3.55 (ABq, 2H, J=14 Hz), 3.57 and 3.37 (ABq, 2H, J=18 Hz), 3.15 (q, 4H,J=7.0 Hz), 1.04 (t, 6H, J=7.0 Hz). ¹³C NMR (500 MHz, CD₃OD): δ 174.6,168.7, 165.3, 136.5, 134.5, 130.2, 129.6, 128.0, 116.9, 74.7, 60.4,58.9, 49.4, 43.1, 26.8, 11.8. IR (cm⁻¹, Neat): 3395, 1765, 1609, 1495,1345, 1248, 997, 679. M.P 35-37° C. [α]_(D) (c=1.0, CH₂Cl₂)=+19.0,ESI-HRMS (m/z) Calcd. for 502.1157 C₂₀H₂₅KN₅O₆S [M+H]⁺, Found 502.1163.

Compound 23

¹H NMR (500 MHz, D₂O): δ 7.35-7.2 (m, 5H), 5.53 (d, 1H, J=4 Hz), 5.05and 4.72 (ABq, 2H, J=12 Hz), 4.99 (d, 1H, J=4 Hz), 3.62-3.58 (m, 2H),3.50 and 3.30 (ABq, 2H, J=13 Hz), 3.4 (m, 4H), 1.82 (m, 4H). ¹³C NMR(125 MHz, D₂O): δ 175.5, 168.4, 164.9, 135.0, 132.3, 129.3, 129.1,127.9, 116.5, 72.9, 59.2, 58.0, 51.6, 42.3, 25.8, 22.5. FTIR (cm⁻¹,Neat): 3399, 3283, 3194, 2976, 2980, 2897, 1758, 1648, 1609, 1542, 1492,1451, 1386, 1351, 1224, 1001. [α]_(D) (c=1.0, MeOH)=−390.3. ESI-HRMS(m/z) Calcd. for 500.1001 [M+H]⁺ C₂₀H₂₃KN₅O₆S⁺, Found 500.1015.

Compound 24

¹H NMR (300 MHz, CD₃OD): δ 8.33 (s, 2H), 7.39-7.26 (m, 5H), 7.32 (d, 1H,J=7.5 Hz), 6.56 (t, 1H, J=2 Hz), 5.63 (d, 1H, J=5 Hz), 5.24 and 4.95(ABq, 2H, J=13.5 Hz), 5.18 (d, 2H, J=2.5 Hz), 4.91 (d, 1H, J=4.5 Hz),3.98 (m, 4H, J=4.5 Hz), 3.66 and 3.61 (ABq, 2H, J=16 Hz), 3.53 and 3.40(ABq, 2H, J=18.5 Hz), 3.41 (t, 4H, J=4.5 Hz). ¹³C NMR (125 MHz, CD₃OD):171.0, 164.7, 161.3, 159.9, 157.8, 133.6, 130.7, 129.4, 129.2, 127.7,126.6, 125.8, 125.7, 113.9, 110.7, 71.8, 68.1, 59.2, 57.4, 55.2, 50.9,43.3, 42.3, 26.1. FTIR (cm⁻¹, Neat): 3394, 3282, 3190, 3034, 2980, 2894,1756, 1645, 1609, 1543, 1492, 1448, 1352, 1224, 994. [α]_(D) (c=1.0,MeOH)=−190.6, ESI-HRMS (m/z) Calcd. for 593.1328 [M+H]⁺ C₂₄H₂₆KN₈O₆S⁺,Found 593.1366.

Diazeniumdiolates 2 and 6 to 8 may be prepared according to literatureprocedures. By way of example, compound 2 was prepared as follows.

(Reference: K. R. A. Abdellatif et al./Bioorg. Med. Chem. 15 (2007)6796-6801).

Diethylamine (7.3 g, 0.1 mol) was added to a solution of NaOMe (0.1 mol,24 mL of a 25% w/v solution in MeOH) and diethyl ether (300 mL) withstirring at 25° C. This mixture was purged with dry argon for 5 min, andthen the reaction was pressurised under an atmosphere of nitric oxide(40 psi internal pressure) with stirring at 25° C. for 5 h. The product,which precipitated as a fine white powder, was isolated by filtration,suspended in diethyl ether (100 mL) and stirred for 15 min. Thesuspension was filtered and the solid collected was dried at 25° C.under reduced pressure until a constant weight was obtained. O²-sodium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) was affordedas a fine white powder (4.0 g, 26%). The product was stored in ambercoloured bottles at −20° C. under argon atmosphere and used withoutfurther purification. ¹H NMR (D₂O) δ 1.12 (t, J=7.3 Hz, 6H, N(CH₂CH₃)₂),2.93 [q, J=7.3 Hz, 4H, N(CH₂CH₃)₂]. m.p. 200-202° C.

Example 2 Nitric Oxide Release from Compounds 14 to 19 In Vitro and byP. Aeruginosa Extracts

Nitric oxide release from compounds 14 to 19 was detected using thenitric oxide specific probe ISO-NOP with an Apollo 4000 analyser (WorldPrecision Instruments). The nitric oxide probe, which was freshlycalibrated using a solution of SNAP according to the manufacturer'sinstructions, was immersed in a vial containing Tris buffer at pH 7.0and continuously stirred at room temperature. Various reagents wereadded successfully while monitoring nitric oxide levels.

Referring to FIG. 1, low μM concentrations of nitric oxide were shown tobe released from all of the cephalosporin-3′-diazeniumdiolate free acids14 to 19 when exposed in aqueous buffer (pH 7) to a commerciallyavailable β-lactamase (penicillinase, Sigma Aldrich). Nitric oxiderelease could not be detected when the enzyme was added withoutcephalosporin-3′-diazeniumdiolate (data not shown). Similarly, whencephalothin alone (closely related to the β-lactam antibiotic backboneof the cephalosporin-3′-diazeniumdiolates) was used instead of acephalosporin-3′-diazeniumdiolate, no release of nitric oxide wasobserved in the presence or absence of penicillinase (data not shown).

The release of nitric oxide by cleavage of compound 15 was studiedfurther using β-lactamase producing P. aeruginosa extracts. P.aerugionsa cells were grown in the presence of subinhibitoryconcentrations of ampicillin (50 μg/ml) to induce β-lactamase activity.β-lactamase producing P. aeruginosa cells were then lysed using CelLyticreagents (Sigma) and cell extracts were added to a 10-ml-solution ofcompound 15 while monitoring nitric oxide production using the nitricoxide electrode. FIG. 2 a shows that release of nitric oxide fromcompound 15 triggered by penicillinase varies slightly between pH 5 and7, while at pH 9 nitric oxide release is greatly reduced. FIG. 2 b showsthat nitric oxide is released from compound 15 following treatment withβ-lactamase expressing P. aeruginosa cell extracts, and that a smalleryet measurable release of nitric oxide from compound 15 occurs upontreatment with non-β-lactamase expressing E. coli cells. This suggeststhat biofilm-forming bacteria that do not express β-lactamase may alsobe induced to disperse by triggering cephalosporin-3′-diazeniumdiolatesto release nitric oxide, possibly via reaction with transpeptidases, theprincipal target of bactericidal cephalosporin antibiotics.

Cephalosporin-3′-diazeniumdiolates may be expected to release nitricoxide better if the biofilm bacteria being targeted secreteextracellular β-lactamases, as opposed to expressing β-lactamases thatare more typically localised in the periplasmic space of cells, which inbiofilms would make the enzymes potentially inaccessible to compounds.FIG. 2 c shows that P. aeruginosa can be induced to expressextracellular β-lactamases that trigger release of nitric oxide fromcompound 15 by pre-treatment with the β-lactam antibiotic imipenem. Thesame induction of extracellular β-lactamase is not apparent with allβ-lactam antibiotics, as evidenced in FIG. 2 c by the failure ofampicillin to induce expression. Thus, imipenem pretreatment affordsextracellular β-lactamase expression, which subsequently leads toincreased nitric oxide release from cephalosporin-3′-diazeniumdiolates.

Example 3 Induction of Nitric Oxide Release from Compound 21 in P.Aeruginosa cells

To study nitric oxide release in intact cells of P. aeruginosa, the P.aeruginosa NSGFP reporter strain was used (Barraud et al., 2009). Thisstrain harbours a gene reporter construct that expresses greenfluorescent protein (GFP) when the nitric oxide-inducible nirS gene isexpressed. P. aeruginosa NSGFP cells were grown with or withoutampicillin at subinhibitory concentration (50 μg/ml) to induceβ-lactamase activity. Aliquots of 3 ml bacterial culture were thenincubated with compound 21, the known nitric oxide donor sodiumnitroprusside (SNP), cephalothin (Sigma ref #C4520) or penicillinase for2 h, before measuring the GFP fluorescence of NSGFP cells.

As shown in FIG. 3 compound 21 (150 μM) alone induced a GFP-responsegreater than the response triggered by SNP in P. aeruginosa cells withor without pre-activation of β-lactamase activity. Moreover, compound 21cleavage and nitric oxide availability to bacteria were enhanced when P.aeruginosa β-lactamase activity was induced by treatment with asubinhibitory concentration of ampicillin. The enhanced response wasequivalent to that observed when compound 21 was co-administered withpenicillinase to cells grown in the absence of ampicillin. Controltreatments that consisted of penicillinase alone or cephalothin had noeffect on GFP fluorescence in the NSGFP reporter assay.

Example 4 Release of Nitric Oxide from Compound 21 Induces BiofilmDispersal

P. aeruginosa PA01 wild type ATCC and MA67 strains, P. aeruginosa FRD1,a mucoid, cystic fibrosis (CF) isolate (Ohman and Chakrabarty, 1981),and the clinical strain P. aeruginosa 18A, which was isolated from CFsputum samples of chronically infected individual in Tasmania (Kirov etal, 2007) were used for biofilm dispersal assays. Biofilms were grown inM9 minimal medium (containing 48 mM Na₂HPO₄, 22 mM KH₂PO₄, 9 mM NaCl, 19mM NH₄Cl, pH 7.2, 2 mM MgSO₄, 20 mM glucose, and 100 μM CaCl₂) in24-well plates batch cultures at 37° C. with shaking at 200 rpm. After 6h incubation for PA01 wild-type biofilms or 24 h incubation for FRD1 and18A biofilms, treatments were added to each well in less than 1 min perplate and the plates were incubated for a further 10 min at 37° C. withshaking at 200 rpm. Planktonic biomass was quantified by directmeasurement of OD₆₀₀ of the supernatant, and the remaining biofilmbiomass was quantified by crystal violet staining (O'Toole and Kolter,1998).

As shown in FIG. 4 Compound 21 (denoted as “DEA-CP”) is potent atinducing dispersal of preestablished biofilms of P. aeruginosa. After 10min exposure, the biofilm biomass, as measured with CV staining, wasreduced by 55%, while the planktonic biomass, as measured bymeasurements of OD_(600nm) of the supernatants, simultaneously increasedby 20% compared to controls that were left untreated (P<0.01, 1-wayANOVA and Tukey's multiple comparison test). In contrast, cephalothinalone did not induce significant dispersal events.

Compound 21 was then tested in a range of concentrations against P.aeruginosa PAO1 wild type biofilms. As shown in FIG. 5 Compound 21(denoted as “DEA-CP”) induces dispersal in a dose dependent manner inthe range 10-100 μM (FIG. 5A). Compound 21 was also tested againstbiofilms of P. aeruginosa CF isolate strains FRD1 and 18A grown for 24h. Exposure to 100 μM Compound 21 for 10 min induced 30% and 35%dispersal in FRD1 and 18A biofilms, respectively, as measured by CVstaining (FIG. 5B).

The dispersal of pre-established P. aeruginosa biofilms (using strainMA67 that forms better biofilms than the strain used in experimentsdescribed above) by compound 21 and its analogue (compound 23) wasfurther assessed and shown to be dose dependent (see FIG. 6).

Further experiments with compound 21 demonstrated the enhancedbiofilm-dispersion effects in P. aeruginosa after induction ofextracellular β-lactamase expression through pre-treatment of biofilmswith imipenem (see FIG. 7). In these experiments, pre-established P.aeruginosa biofilms were grown in microtiter plates with shaking at 37°C. and treated with compound 21 at 10 μM and 100 μM in the presence orabsence of penicillinase (0.1 U/mL) for 15 min or left untreated.Pre-established P. aeruginosa that were pre-treated with imipenem at 0.5μg/mL for 1 h to induce release of extracellular β-lactamase were thenexposed to compound 21 at 10 μM and 100 μM or 15 min.

The inventors then investigated the ability of compound 21 to inducedispersal of biofilms from other Gram negative bacterial species(Escherichia coli, Vibrio cholerae and Serratia marcescens) and the Grampositive species Staphylococcus aureus. As shown in FIG. 8, compound 21induces rapid dispersal (10 min after treatment) in biofilms of thesespecies.

The biofilm dispersing properties of compound 21 were furtherinvestigated using continuous-flow biofilm culture assays. P. aeruginosaPAO1 biofilms were established in glass microfermenters receiving acontinuous flow of fresh M9 minimal medium. The inlet was switched tovessels containing fresh medium, with or without compound 21 (100 μM),and OD₆₀₀ measurements of the effluent taken. A rapid and significantincrease in released cells was observed after addition of compound 21,while the amount of cells released from untreated biofilms remainedunchanged (FIG. 9).

Example 5 Minimum Inhibitory Concentration of Compound 21

P. aeruginosa PA01 wild type cells were grown in M9 minimal medium in96-well plates at 37° C. under static conditions in the presence of arange of concentrations of Compound 21 diethylamine diazeniumdiolate(DEA; nitric oxide donor used for synthesis of Compound 21), and/or theantibiotics cephalothin, ceftazidime and tetracycline. Planktonic growthwas quantified at various time points by direct measurement of OD₆₀₀ ofthe supernatant. The minimum inhibitory concentration (MIC) forantibiotic treatments was defined as the concentration of antibioticthat resulted in an OD_(600nm) of 35% or less after 20 h growth. Forbiofilm viability experiments, P. aerugionsa biofilms were grown in24-well microtitre plates as described above (see Example 4) for 7 h,before adding treatments of Compound 21 and antibiotics at the indicatedfinal concentrations and incubating for another 1 h or 2 h under normalculture conditions. For each time point, planktonic bacteria werecollected, serially diluted in sterile phosphate-buffered saline (PBS)and spread on LB agar plates. Biofilms were gently rinsed once with PBSand resuspended from the wall and bottom well surfaces (total surfacearea=4.4 cm²) using a sterile cotton swab. Bacterial aggregates werefurther disrupted in a bath sonicator for 10 min. Biofilm bacteria werethen serially diluted and spread on LB agar plates. Colony forming units(CFU) were determined after 1 day incubation at 37° C.

Compound 21 alone was found to be more potent at inhibiting P.aeruginosa planktonic growth with a MIC of 4 mM compared to cephalothinor DEA, which were both found to have an MIC of 16 mM (FIG. 10—Compound21 denoted as “DEA-CP”). The growth inhibitory effects of Compound 21are likely due to toxic levels of nitric oxide, and its increasedtoxicity compared to DEA is suggested to be the result of targetedrelease of nitric oxide within bacteria.

Example 6 Combinatorial Treatments Involving Compound 21 and Antibiotics

To assess the effect of Compound 21 and antibiotics on the viability ofbiofilm and dispersed cells, combinatorial treatments of Compound 21with ceftazidime were carried out on biofilms grown in microtitre plates(as described above in Example 5), and both biofilm CFU and planktonicCFU were determined The results demonstrate a concomitant decrease inbiofilm CFU, from 1.9×10⁸ to 1.3×10⁸ CFU per well, and increase inplanktonic CFU, from 4.8×10⁸ to 11×10⁸ CFU per well, after 1 h treatmentwith Compound 21 100 μM alone (see FIGS. 11A and 11B), which isconsistent with OD and CV measurements previously obtained (see Example4; FIG. 4). Further, the efficacy of 10 μM ceftazidime against bothbiofilm and planktonic bacteria was increased when incubated in thepresence of Compound 21 for 1 h compared to biofilm and planktonicbacteria that were treated with ceftazidime alone (FIGS. 8A and 8B). Theefficacy of ceftazidime against biofilm bacteria was also increased by2.3-fold in the presence of Compound 21 after 2 h treatments compared tobiofilms that were treated without Compound 21 (FIG. 11C).

The inventors further found that compound 21 potentiates theanti-biofilm efficacy of the clinically employed antibiotics tobramycinand ciprofloxacin (Table 1). Established P. aeruginosa biofilms grown inmicrotiter plates with shaking at 37° C. were pre-treated withsub-inhibitory imipenem (10 μg/mL) for 1 h to induce release ofextracellular β-lactamase. Tobramycin or ciprofloxacin were then addedeither with or without compound 21, or biofilms were left untreated(controls). Biofilm bacteria were incubated for a further 1 h beforeresuspension and enumeration of cfu. Values represent the log-foldreductions in cfu compared to control biofilms and are the mean of twoindependent experiments (±SEM). Bacteria in the supernatants were fullyeradicated (below the detection limit; 10 cfu/mL) after treatment witheither antibiotic alone or either antibiotic in combination withcompound 21.

TABLE 1 Compound 21 Log Reduction Antibiotic (μM) in Biofilm cfuTobramycin 0 2.15 ± 0.16 80 μg/mL/170 μM 10 3.92 ± 0.06 100 3.58 ± 0.15Ciprofloxacin 0 3.54 ± 0.09 5 μg/mL/15 μM 10 5.06 ± 0.02 100 4.90 ± 0.13

In combinational treatments with tetracycline, against planktonic P.aeruginosa Compound 21 increased the inhibitory effects of 6 μMtetracycline by 45% and the inhibitory effects of 25 μM tetracycline by60% (data not shown).

REFERENCES

Barraud N, et al. (2009) Nitric oxide signaling in Pseudomonasaeruginosa biofilms mediates phosphodiesterase activity, decreasedcyclic di-GMP levels, and enhanced dispersal. J. Bacteriol.191(23):7333-7342.

Hope et al, 2002, Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viabilityprofiling, J. Appl. Microbiol., 1993: 448-455.

Kirov S M, et al. (2007) Biofilm differentiation and dispersal in mucoidPseudomonas aeruginosa isolates from patients with cystic fibrosis.Microbiology 153(Pt 10):3264-3274.

Ohman D E & Chakrabarty A M (1981) Genetic mapping of chromosomaldeterminants for the production of the exopolysaccharide alginate in aPseudomonas aeruginosa cystic fibrosis isolate. Infect. Immun.33(1):142-148.

O'Toole G A & Kolter R (1998) Initiation of biofilm formation inPseudomonas fluorescens WCS365 proceeds via multiple, convergentsignalling pathways: a genetic analysis. Mol. Microbiol. 28(3):449-461.

Webb et al, 2003, Cell death in Pseudomonas aeruginosa biofilmdevelopment, J. Bact., 185: 4585-4592.

1. A compound of the formula (I), or a salt thereof:

wherein T is a bond or a linker, R₂ and R₃ are organic residues and X isselected from the group consisting of:

wherein R₁ is an organic residue.
 2. A compound according to claim 1having the structure:

wherein R₁, R₂, R₃ and T are as defined above, including salts thereof3. A compound according to claim 1 or claim 2 wherein T is a linkerwhich is a bivalent hydrocarbon having between 1 and 6 carbon atoms. 4.A compound according to claim 3 wherein T is CH₂.
 5. A compoundaccording to any one of claims 1 to 4 wherein R₁ is a substituent thatcorresponds to a substituent attached to the 7-NHC(O)— group of acephalosporin antibiotic.
 6. A compound according to any one of claims 1to 5, wherein R₁ is selected from the group consisting of:


7. A compound according to any one of claims 1 to 4 wherein R₁ is Y-arylor Y-heteroaryl where Y is a bivalent hydrocarbon having between 1 and 4carbon atoms.
 8. A compound according to claim 7 wherein aryl isselected from the group consisting of: phenyl, biphenyl, naphthyl,anthracenyl and phenanthrenyl, and wherein heteroaryl is a 5- or6-membered ring wherein between 1 and 4 carbon atoms are replaced withnitrogen and/or sulfur atoms.
 9. A compound according to claim 7 orclaim 8 wherein heteroaryl is selected from the group consisting of:thienyl, tetrazolyl, imidazolyl, triazolyl and pyrrolyl.
 10. A compoundaccording to claim 7 wherein R₁ is selected from the group consistingof: —CH₂-phenyl, —CH₂-thienyl and —CH₂-tetrazolyl.
 11. A compoundaccording to any one of claims 1 to 10 wherein R₂ and R₃ areindependently =selected from: hydrogen, C₃-C₇ cycloalkyl, C₃-C₇cycloalkenyl, (CH₂)_(p)OC(O)PhOC(O)C₁-C₆alkyl,(CH₂)_(p)OC(O)APhC₁-C₆alkyl, branched or straight chain C₁-C₂₀ alkyl,branched or straight chain C₂-C₂₀ alkenyl, branched or straight chainC₂-C₂₀ alkynyl, wherein the alkyl, alkenyl or alkynyl chains mayoptionally be interrupted by one or more groups/heteroatoms selectedfrom O, S, NH, NH₂ ⁺, and wherein the alkyl, alkenyl or alkynyl groupsmay optionally be substituted by one or more substituents selected fromthe group consisting of: halogen, cyano, COOH, (CH₂)_(p)C(O)OC₁-C₆alkyl,C(O)OC₁-C₆alkenyl, SO₃H, SO₂halogen, SO₂NH₂, NH₂, NH₃ ⁺, OH, SH,OC₁-C₆alkyl, OC₂-C₆alkenyl, OC₂-C₆alkynyl, aryl and heteroaryl, oralternatively R₂ and R₃, together with the nitrogen to which they areattached, form a 4-, 5-, 6-, 7-or 8-membered ring which may optionallycontain 1, 2, 3, 4, 5 or 6 additional nitrogen atoms and may besaturated, unsaturated or partially unsaturated, and wherein the 4-, 5-,6-, 7-or 8-membered ring may optionally be substituted by one or moresubstituents selected from the group consisting of:—C(O)C₁-C₃alkylene-naphthyl-OC₁-C₆alkyl, —C(O)C₁-C₃alkylene-Ph—C(O)—Ph,—C(O)CH₂O(CH₂)_(p)OCH₃, —C(O)OPhNO₂, —C(O)OPhNH₂,—C(O)O(CH₂)_(p)Chalogen₃, —C(O)O(CH₂O)_(p)CH₃, C₁-C₆-alkyl,C₂-C₆alkenyl, C₂-C₆alkynyl, —C(O)C₁-C₆alkyleneCOOC₁-C₆alkyl,—C(O)C₁-C₃alkylenePhC₁-C₆alkyl, —C(O)O-pyrrolidinyl-2,5-dione,—C(O)C₁-C₃alkylenePhC₁-C₆alkyl, —C(O)(CH₂)_(p)OC₁-C₆alkyl,—C(O)O(CH₂)_(p)halogen, —C(O)O(CH₂)_(p)Ph, —(CH₂)_(p)SH,-S0₂naphthyl-NC₁-C₆alkyl, —C(O)ONC₁-C₆alkyl, —(CH₂)_(p)OH, —C(O)PhOAc,—C(O)(CH₂)_(p)NHC(O)C₁-C₆alkyl, —C(O)NH₂, —C(O)M, —C(O)NR₄R₅,—(CH₂)_(p)CH(OH)CHOH, halogen, cyano, —COOH, —C(O)O(CH₂)_(p)Ph,—C(O)OC₁-C₆alkyl, —C(O)OC₂-C₆alkenyl, —C(O)OC₂-C₆alkynyl,—C(O)SC₁-C₆alkyl, —C(O)SC₂-C₆alkenyl, —C(O)SC₂-C₆alkynyl,—C(O)C₁-C₆alkyl, SO₃H, SO₂halogen, SO₂phenyl, SO₂NH₂, SO₂NR₄R₅,SO₂PhNHCOC₁-C₆alkyl, NH₂, OH, SH, OC₁-C₆alkyl, OC₂-C₆alkenyl,OC₂-C₆alkynyl, aryl and heteroaryl, and wherein A is a bivalenthydrocarbon radical having between 1 and 4 carbon atoms, p is a numberbetween 0 and 4, R₄ and R₅ independently represent C₁-C₆alkyl and M ispyridyl, pyrimidinyl, pyrazinyl, phenyl or triazinyl.
 12. A compoundaccording to any one of claims 1 to 10 wherein R₂ and R₃ areindependently selected from: hydrogen, C₅-C₇ cycloalkyl,(CH₂)_(p)OC(O)PhOC(O)C₁-C₆alkyl, (CH₂)_(p)OC(O)APhC₁-C₆alkyl, branchedor straight chain C₁-C₁₀ alkyl, branched or straight chain C₂-C₁₀alkenyl and branched or straight chain C₂-C₁₀ alkynyl, wherein thealkyl, alkenyl or alkynyl chains may optionally be interrupted bybetween one and three groups/heteroatoms selected from O, S, NH and NH₂⁺, and wherein the alkyl, alkenyl or alkynyl chains may optionally besubstituted by between one and six substituents selected from the groupconsisting of: halogen, phenyl, ethoxy, methoxy, propoxy, COOH,(CH₂)_(p)COOC₁-C₄alkyl, NH₂, NH₃ ⁺, OH and SH, and wherein A is abivalent hydrocarbon radical having between 1 or 2 carbon atoms and p is0, 1 or
 2. 13. A compound according to any one of claims 1 to 10 whereinR₂ and R₃ are independently selected from: hydrogen, C₅-C₇ cycloalkyl,branched or straight chain C₁-C₁₀ alkyl, branched or straight chainC₂-C₁₀ alkenyl, branched or straight chain C₂-C₁₀ alkynyl, wherein thealkyl, alkenyl or alkynyl chains may optionally be interrupted bybetween one and three groups selected from O, NH and NH₂ ⁺, and whereinthe alkyl, alkenyl or alkynyl chains may optionally be substituted bybetween one and four substituents selected from the group consisting of:halogen, phenyl, methoxy, COOH, CH₂COOC₁-C₄alkyl, NH₂ and NH₃ ⁺.
 14. Acompound according to any one of claims 1 to 10 wherein R₂ and R₃ areindependently selected from: hydrogen, cyclohexyl, branched or straightchain C₁-C₁₀ alkyl or branched or straight chain C₂-C₁₀ alkenyl, whereinthe alkyl or alkenyl chains may optionally be interrupted by one or twogroups selected from NH and NH₂ ⁺, and wherein the alkyl, alkenyl oralkynyl chains may optionally be substituted by between one and threesubstituents selected from the group consisting of: phenyl, methoxy,COOH, NH₂ and NH₃ ⁺.
 15. A compound according to any one of claims 1 to10 wherein R₂ and R₃, together with the nitrogen to which they areattached, form a 4-, 5-, 6- or 7-membered ring which may optionallycontain 1, 2, 3, 4 or 5 additional nitrogen atoms and may be saturated,unsaturated or partially unsaturated, and wherein the 4-, 5-, 6- or7-membered ring may optionally be substituted by one or moresubstituents selected from the group consisting of: SO₂NMe₂, SO₃H,SO₂halogen, SO₂NH₂, —C(O)O(CH₂)_(p)Ph, —C(O)Me, —C(O)pyridyl,—(CH₂)_(p)OH, —C(O)NH₂, —COOH, —C(O)NMe₂, —C(O)NEt₂, phenyl, naphthyl,pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolidinyl, imidazolyl,C₁-C₆-alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, —C(O)OC₁-C₆alkyl,—C(O)OC₂-C₆alkenyl, —C(O)OC₂-C₆alkynyl, —C(O)O(CH₂)_(p)Ph, —(CH₂)_(p)SH,halogen, SO₂PhNHCOC₁-C₆alkyl, NH₂, SH, OC₁-C₆alkyl, and wherein p is anumber between 0 and
 2. 16. A compound according to any one of claims 1to 10 wherein R₂ and R₃, together with the nitrogen to which they areattached, form a 5-, 6-, or 7-membered ring which may optionally contain1, 2 or 3 additional nitrogen atoms and may be saturated, unsaturated orpartially unsaturated, and wherein the 5-, 6-, or 7-membered ring mayoptionally be substituted by between one and four substituents selectedfrom the group consisting of: SO₂NMe₂, SO₂NH₂, —COO(CH₂)_(p)Ph —C(O)Me,—C(O)pyridyl, —(CH₂)_(p)OH, —C(O)NH₂, —COOH, —C(O)NMe₂, —C(O)NEt₂,phenyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolidinyl,imidazolyl, C₁-C₆-alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, —C(O)OC₁-C₆alkyl,—C(O)OC₂-C₆alkenyl, —C(O)OC₂-C₆alkynyl, —C(O)O(CH₂)_(p)Ph, —(CH₂)_(p)SH,halogen, NH₂, SH, OC₁-C₆alkyl, p is a number between 0 and
 2. 17. Acompound according to any one of claims 1 to 10 wherein R₂ and R₃,together with the nitrogen to which they are attached, form a saturated5-, 6-, or 7-membered ring which may optionally contain 1, 2 or 3additional nitrogen atoms, and wherein the 5-, 6-, or 7-membered ringmay optionally be substituted by between one and three substituentsselected from the group consisting of: SO₂NMe₂, SO₂NH₂, —C(O)Me,—C(O)pyridyl, —(CH₂)_(p)0H, —C(O)NH₂, —COOH, —C(O)NMe₂, —C(O)NEt₂,phenyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolidinyl,imidazolyl, C₁-C₆-alkyl, C₂-C₆alkenyl, —C(O)OC₁-C₆alkyl,—C(O)OC₂-C₆alkenyl, halogen, NH₂, SH, OC₁-C₆alkyl, p is a number between0 and
 2. 18. A compound according to any one of claims 1 to 10 whereinR₂ and R₃, together with the nitrogen to which they are attached form astructure selected from the group consisting of:


19. A compound according to any one of claims 1 to 10 wherein R₂ and R₃are independently selected from C₁-C₁₀ alkyl, or alternatively R₂ andR₃, together with the nitrogen to which they are attached, form a 5- or6-membered ring which may optionally contain between 1 and 3 additionalnitrogen atoms, and which may optionally be substituted with an aryl orheteroaryl group.
 20. A compound according to claim 19 wherein R₂ and R₃are independently selected from C₁-C₆ alkyl, or alternatively R₂ and R₃,together with the nitrogen to which they are attached, form a saturated5- or 6-membered ring which may optionally contain 1 additional nitrogenatom, and which may optionally be substituted with a substituentselected from the group consisting of: pyrimidinyl and phenyl.
 21. Acompound according to claim 20 wherein R₂ and R₃ are independentlyselected from C₁-C₆ alkyl, or alternatively R₂ and R₃, together with thenitrogen to which they are attached, form a structure selected from thegroup consisting of:


22. A compound according to claim 1 or 2 having a structure selectedfrom the group consisting of:

including salts thereof.
 23. A compound according to any one of claims 1to 22 further comprising an antibiotic compound attached via the R₂and/or R₃ substituent.
 24. A compound according to claim 23 wherein theantibiotic is ciprofloxacin or N-desmethyl levofloxacin.
 25. Acomposition for promoting the dispersal of microorganisms from a biofilmor inhibiting the formation and/or development of biofilms, thecomposition comprising a compound according to any one of claims 1 to24.
 26. A composition according to claim 25 further comprising one ormore additional antibiotics or antimicrobial agents.
 27. A compositionaccording to claim 25 or 26 wherein the composition inducesdifferentiation events in microorganisms within biofilms which lead todispersal.
 28. A composition according to claim 26 or 27 wherein thecomposition prevents induction of differentiation events inmicroorganisms which lead to biofilm formation.
 29. A compositionaccording to any one of claims 25 to 28 wherein the compositionincreases the sensitivity of biofilm-forming microorganisms to anantimicrobial agent or antibiotic.
 30. A method for promoting dispersalof microorganisms from a biofilm, the method comprising exposing thebiofilm to an effective amount of a compound according to any one ofclaims 1 to 24 or a composition according to any one of claims 25 to 29.31. A method for inhibiting biofilm formation and/or development, themethod comprising exposing biofilm-forming microorganisms to aneffective amount of a compound according to any one of claims 1 to 24 ora composition according to any one of claims 25 to
 29. 32. A methodaccording to claim 31 wherein the compound or composition is coated,impregnated or otherwise contacted with a surface or interfacesusceptible to biofilm formation.
 33. A method according to claim 32wherein the surface is a surface of an implantable medical device,prosthesis or medical or surgical equipment.
 34. A method according toany one of claims 30 to 33 wherein the biofilm-forming microorganismsexpress a β-lactamase or a transpeptidase.
 35. A method according toclaim 34 wherein the biofilm-forming microorganisms express aβ-lactamase.
 36. A method according to claim 35 wherein the β-lactamaseis a penicillinase.
 37. A method according to claim 35 or 36 wherein thebiofilm or biofilm-forming microorganisms are exposed to a β-lactamantibiotic prior to or concomitant with exposure to the compound orcomposition.
 38. A method according to claim 37 wherein the β-lactamantibiotic induces production of extracellular β-lactamase in saidbiofilm-forming microorganisms.
 39. A method according to claim 37 orclaim 38 wherein the β-lactam antibiotic is imipenem.
 40. A methodaccording to any one of claims 30 to 39 wherein the biofilm forms on abodily surface of a subject, internal or external to the subject, andexposure of the biofilm or biofilm-forming microorganisms to thecompound or composition is via administration of the compound orcomposition to the subject.
 41. A method according to any one of claims30 to 40 wherein promoting dispersal of microorganisms from a biofilm orpreventing formation of biofilms comprises inducing differentiationevents in microorganisms within biofilms which lead to dispersal orcomprises preventing induction of differentiation events inmicroorganisms which lead to biofilm formation.
 42. A method accordingto any one of claims 30 to 40 wherein promoting dispersal ofmicroorganisms from a biofilm or preventing formation of biofilmscomprises increasing the sensitivity of a microorganism to antimicrobialagents.
 43. A method according to any one of claims 30 to 42 wherein thebiofilm or biofilm-forming microorganisms are exposed to an effectiveamount of the compound or composition such that the concentration of thenitric oxide donor or nitric oxide released and thus exposed to thebiofilm or microorganisms is non-toxic to the environment or to thesubject in which the biofilm or microrgansims are found.
 44. A methodaccording to claim 43 wherein the concentration of nitric oxide is inthe nanomolar, micromolar or millimolar range.
 45. A method according toclaim 44 wherein the nitric oxide concentration is from about 1 nM toabout 500 μM.
 46. A method according to any one of claims 30 to 45wherein the biofilm is a single species biofilm.
 47. A method accordingto any one of claims 30 to 45 wherein the biofilm is a multiple speciesbiofilm.
 48. A method according to any one of claims 30 to 47 whereinthe microorganisms within the biofilm or capable of forming a biofilmcomprise one or more species selected from Pseudomonas spp.,Pseudoalieromonas spp., Staphylococcus spp,., Streptococcus spp.,Shigella spp., Mycobacterium spp., Enterococcus spp., Escherichia spp.,Salmonella spp., Legionella spp., Haemophilus spp., Bacillus spp.,Desulfovibrio spp., Shewanella spp., Geobacter spp., Klebsiella spp.,Proteus spp., Aeromonas spp., Arthrobacter spp., Micrococcus spp.,Burkholderia spp., Serratia spp., Porphyromonas spp., Fusobacterium spp.and Vibrio spp.
 49. A method according to claim 48 wherein themicroorganisms are selected from Pseudomonas aeruginosa, Staphylococcusepidermidis, Staphylococcus aureus, Mycobacterium tuberculosis,Escherichia coli, Bacillus licheniformis, Burkholderia cenocepacia,Serratia marcescens, Fusobacterium nucleatum, and Vibrio cholerae.
 50. Amethod according to any one of claims 30 to 49 wherein the biofilm is onor within the body of a subject and is associated with a disease ordisorder suffered by the subject.
 51. A method according to claim 50wherein the disease or disorder is selected from cystic fibrosis,bacterial endocarditis, otitis media, Legionnaire's disease,tuberculosis and kidney stones.
 52. A method for treating or preventinga biofilm-associated infection, disease or disorder in a subject whereinthe infection is caused by a microorganism capable of forming a biofilm,the method comprising administering to the subject an effective amountof a compound according to any one of claims 1 to 24 or a compositionaccording to any one of claims 25 to
 29. 53. Use of a compound accordingto any one of claims 1 to 24 for the manufacture of a composition foruse in promoting dispersal of microorganisms from a biofilm or forinhibiting the formation or development of a biofilm.
 54. Use of acompound according to any one of claims 1 to 24 for the manufacture of amedicament for treating or preventing a biofilm-associated infection,disease or disorder in a subject wherein the infection is caused by amicroorganism capable of forming a biofilm.